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LUMINESCENCE ACTIVITY AS A BIOSENSOR OF

1. n k a snp AE SE SIS SWE Ed St J 42 MOU JOJ 22 sdojs uomnezi eriut OY o SUIUINJOI spuo 4007 suissoooug uonismboy v payoeal SI uonIsod o qe3uunj MOU v UYM PALS pue pear sr 29 Iy pue p ddo s ST e3oureo oq O QP IBAR SI gp oSeurr pue p p x 994 seq own msodx v uo D ApIdem pue poje no eo st oum oq doo Suiss ooiq uonrsimbov n UYUM msodx oY Surejs pue PIOUS ou Suumsuuoo doo 93PUI urew n ur INDIO sdojs uonegnrur 201 mp ooid uonisinb c ogeu Surordop wergerp y olq orAqe CN IANS sa pueH uogsinbay d abeuur pur ajg L ajpueH aid 3 puey sawe a puer ewe ssppueH 2320 say aul 11535 ainsodx3 E K 1 BT e KE e H a m d D BTB d aaa E 5 1 E D 2 beuu PUUE DDPA 7 2 2 ye beu uonsinboy uie d 3uauuuadx3 e ber Yessy JO dxqmapy d d H H l D D D d H l H D D D d H H D D H d d H l H D D d d H l D D d d H H H D H D D cu 51312 UU e uansinb3y aula suon un4 dao buissa2o1g uonsinbay doo beu au pez 3usuuadx34 amp 43 P APS Auo o2 pue suormeoo Kuo oo s1ojoure1ed Su
2. YY at SUL JLEM ou HUE PISLEU JOU ugu AN js u Je UOTSOd tuo l o JUAS 51 oq umisoid uonopuos p ypuronr Jo suonoouuoo o1eA3jos 38unortd p yeyo MO EZ IANS sb p x4 oul HEM isnieis ejeq a3eu ane AISNVd B s eg S A ejeq spul u 19J ageul SS 32O1d 0011504 1X N o 9 qequun puas uonisinboy 938141 3181114 SUuISS 2O1d 93841 9181114 ejeq SUISSIIOJd uonisog JWOH s un3 s 149 41 0 0 9 qequun pues 9 N3IJUO WeJSOJd S A dois WeJ1301d UOI NAY 26 Image Acquisition Images are acquired using the Scientific Imaging ToolKit SITK software package which is commercially available RCubed Software and run through LabView VIs The SITK package employs pvcam software designed for Princeton Instruments cameras to send commande to the PIXIS 512 camera The SITK software Is configured to initialize the camera check the camera temperature set the exposure time control shutter functioning perform online background subtraction and save the resulting image Under the Single Image tab the user can select whether or not acquire and subtract a background image When the Ktron Program Is running a background image 1s taken at the beginning of each cycle with the SITK software ensuring the camera shutter remains closed for the duration of the exposure The software then s
3. S TUO7O2 YIM S uSIp 1124 251 S HSIp JO soje d ploy S 9UURBYO 21 genue yq 12M 1OO P916o6o 22 o11s0dxo joureo SULINP SSIUNIBP ur s us p ou sd ysuL AO UOIPIODIR MIBTQ o IqA sousrp LIA ojeurumq tr o FUMIO DY ZULY SISTI SISP OY ouueuo y ur p uonisod are s us p INdg ujeouoq 1070W 1odd 1s Ota UMOYS SI 3 Q8 U M Y UOnopuoy Jo uonegnsrquoo o1eApaeu Sunord p 501004 Tz IANSIH 3 I A 1 o qeyuun L a v e df n pou Gs qeu S TUO7O2 YIM S uSIp 1124 1981 0 soje d osodx T sduie T 80100 SSIUNIBP ur sojejd 54 SULIDAO O NIP S uSIp JO soje d ploy S SUURYO 21 enue C OO7P9 09 2 automated acquisition series program via a unique user Interface Motor Control Serial communication between the computer and the EMP400 makes use of National Instrument e VISA Virtual Instrument Software Architecture The software consists of two additional options Independent of the automated series The Focus tab allows the user to continuously capture images outputting them In a fraction of a second This feature allows for the user to properly focus the camera when making adjustments to the camera lens or distance between the camera and the turntable The Single Image tab is capable of capturing an image saving an image at a specific location and opening a previously saved Image The feature 1s useful
4. Liu X Brune D Vermaas W amp Curtiss R L 2010 Production and secretion of fatty acids in genetically engineered cyanobacteria PNAS 1 6 Liu Y Golden S Kondo T Ishiura M amp Johnson C 1995 Bacterial luciferase as a reporter of circadian gene expression in cyanobacteria J Bacteriol 177 8 2080 2086 McKinlay J B amp Harwood C S 2010 Photobiological production of hydrogen gas as a biofuel Current Opinion in Biotechnology 21 244 251 Millar A J Carre I A Strayer C A Chua N H amp Kay S A 1995 February 19 Circadian Clock Mutants in Arabidopsis Idenitfied by Luciferase Science 267 1161 1163 Minak Bernero V Bare R E Haith C E amp Grossman M J 2004 July Detection of Alkanes Alcohols and Aldehydes Using Bioluminescence Biotechnology and Bioengineering 87 2 170 177 Mori T amp Johnson C H 2001 Circadian programming in cyanobacteria Cell amp Developmental Biology 12 271 278 Ouyang Y Andersson C Kondo T Golden S amp Johnson C 1998 Resonating circadian clocks enhance fitness In cyanobacteria Proc Natl Acad Sci USA 95 8660 8664 Pimentel D amp Patzek T W 2005 Ethanol Production Using Corn Switchgrass and Wood Biodiesel Production Using Soybean and Sunflower In Biofuels Solar and Wind as Renewable Energy Systems pp 373 394 Ragauskas A J Williams C K Davison B H Britovsek G
5. 20 Flow chart depicting image processing Drocedure 30 15 minute exposures of trcp luxAB at eight different IBA concentrations 34 trcp luxAB cyanobacteria colonies with 5 I8A 2 35 psbAl luxAB cyanobacteria colonies with 5 IBA 36 Labview block diagram depicting software connections of new proeram 41 Labview block diagram depicting automated Kondotron proeram 42 Labview block diagram depicting image acquisition procedure 43 Labview block diagram depicting overview of image processing procedure 44 Labview block diagram depicting details of image processing 45 IN CHAPTER I INTRODUCTION The Growing Importance of Biofuels The worldwide energy consumption rate 1s proyected to Increase 49 by 2035 To date the world s increasing energy demands have been met primarily by fossil fuels 1 e coal oil natural gas However concerns about climate change fossil fuel price increases security of oil supply and depleting reserves of easily extractable oil direct efforts toward implementation of sustainable energy practices Energy Information Administration 2010 Novel technologies utilize the earth s natural resources such as wind ocean tides sunlight and geothermal heat as renewable sources of energy Still liquid fuel remains the primary fuel source of the
6. gt If Dilate is applied to an image that has been thresholded manually it is often necessary to increase the Min value of that threshold Open performs an erosion followed by a dilation Because the erosion is first it functions to remove small groups of selected pixels that are rather isolated from other selected pixels The dilation is only performed on the selected pixels that remain after the erosion Close performs a dilation followed by an erosion Because the dilation is first it functions to fill small holes within a single colony or merge two separate colonies into one It then applies an erosion to make the resultant cluster of pixels smaller by decreasing the area along its boundary Open amp Close uses dual combinations of Open and Close This function calls several combinations of erosions and dilations thereby smoothing colony boundaries Sometimes this function can result in colony boundaries that are too smoothed out and no longer properly fit the shape of the actual colony Colony Separation 1s applied after all morphologies Separation via Watershed applies a filter based on the original image to the binary image The original image is partitioned based on its topographical surface to create a filter Image separated into non overlapping segments When the filter 1s applied to the binary Image selected pixels are divided into separate colonies according to the boundaries of the segments 54 Alt
7. Cairney J Eckert C A et al 2006 January The Path Forward for Biofuels and Biomaterials Science 311 484 489 Schirmer A Rude M A Popova E amp del Cardayre S B 2010 July Microbial Biosynthesis of Alkanes Science 329 559 562 Searchinger T Heimlick R Houghton R A Dong F Elobeid A Fabiosa J et al 2008 February Use of U S Croplands for Biofuels Increases Greenhouse Gases Through Emission from Land Use Change Science 519 1238 1240 Service R F 2009 October Another Biofuels Drawback The Demand for Irrigation Science 326 516 517 62 Service R F 2010 August Is There a Road Ahead for Cellulosic Ethanol Science 329 784 785 Sheehan J 2009 December Engineering direct conversion of CO2 to biofuel Nature Biotechnology 27 12 1128 1129 Somerville C Youngs H Taylor C Davis S C amp Long S P 2010 August Feedstocks for Lignocellulosic Biofuels Science 329 790 792 Stephanopoulos G 2007 Challenges in engineering microbes for biofuels production Science 315 801 804 Stephens E Ross I L King Z Mussgnug J H Kruse O Posten C et al 2010 February An economic and technical evaluation of microalgal biofuels Nature Biotechnology 28 2 126 128 Taylor W Wilson S Presswood R amp Hastings J 1982 Circadian Rhythm Data Collection with the Apple II Microcomputer Journal of Interdisciplinary Cycl
8. e g 180 for 3 min 2 Start Time in hours for the starting time of the 1 data recording point in constant light e g 3 5 hrs 3 Threshold value There are three options from the pull down menu 1 Automatic for automatic selection 11 Manual Dim for dimer colonies min 20 max 20 000 iii Manual Bright for bighter colonies min 200 max 20 000 4 Size for colony size selections from the pull down menu 48 1 Small colonies 11 Medium colonies 111 Large colonies 5 Contrast Brightness for colonies image appearances Note this setting will not affect data e collection and values 6 Sample notes for each channel for your own record Note Changing the threshold type or colony size will automatically change the associated min and max values To make settings the same for all channels tick the box located above each parameter to have all values the same as Ch 1 Un tick this box to make adjustments to channel parameter values 12 Push RUN button to start the JKtron running program 13 Note Motor will go the Home position If motor was previously moving it will stop and then go to the Home position The position indicator will light up once the Home position has been reached O 3 0 nO m D Control panel will be disabled until background 0 image and Ch 0 image are completed s _ Once the motor has moved to Ch 1 the image for
9. 1070W pue eJoureo v 92u ure1804d MOU Jo suonoouuoo o1eA3jos Sunord p WeISeIP YOOTG A orAqe EV m r P uuuuunununun A a gequun al 1 eiauuep 321 2140 31n3e1aduus jar 41 payoeay 401150 a qequun ainje1aduua vm 7 YL a uamnp auua pog 2135 452142 JOJO dog puueyo ueas days atuori uorpa1g paads A unnuununnunn ageun anrop 2 doo uleyy PUUL 1x u v o SIAQUI 38 oq PIPIDIXI 51 SUIT PEM OY pue posned Jou UAM doo Sutss oooiq uonrsinbov ou UIYHM A SNO3UB NUUIIS Inooo SurssoooJd pue vuonisinb r 93801 doot 89011 94 ur pojonpuoo Surss ooid pue vonisinb r Jo uonenru 1 218 20 Je pomsruoo sgunjes pue uonisod 9000 0 1095 sr qgJuin OY ure13o4d uonopuos pojeurojne Sunord p Wieiselp xoo q Morge Z V 9ANSIH L puuey T N u E d x dolar ar n x emer vr Errrrrerni ov ajgequiny 2 531 A 53 puey mg about exauue 2 ERR 2 ESSO A uanisog Gar J E Gas A a a o 3 gun JE qua Lua dx3 3p siu 5p1005 52 40 05 1 burss3301g about 378140 UOIHSO y 94 033 q83u1n EE 5 3j2LUEIE g 9 EJE 3AE 5 souon auupu Buissa301q Duissa301q uonsinb3y dao sbeu M
10. 23ug1ig ddy Auojo5 ei i 2 NE P i TUNA rr IIIIITTTIITTTIZIIIITTTTTTTTTTTTITITITITITTTTTTTTTTITITIITITTTTTTTTTTTTTITITTITTTTTTTT EET EMEN Al SU 31 Li L LU l LI l J a qUOIPSO 3500A 1551 45 APPENDIX B USER MANUAL FOR OPERATION OF PROGRAM Introduction Following is an instruction manual for both new and veteran users of the Kondotron program This manual was written by Yao Xu with modifications by Carl Johnson and Jill Mecklenborg The Johnson group has developed a custom high throughput screening system with a turntable and CCD camera This apparatus can distinguish as many as 1 000 colonies per plate and has the capacity to automatically monitor 12 plates Therefore 12 000 colonies can be screened or monitored each time The Johnson lab has successfully applied this automated apparatus to isolate and characterize a variety of clock mutants in cyanobacteria Operational Manual Before Using JKtron e Check waterbath s water level If the level is low please refill waterbath Miliipore water before AND during experiment running whenever the level is low Check UPS if the battery is low or dead DLE Clean turning table 0 Keep the JKtron room clean Complete JKtron experiment worksheet see the last page of manual Temperature p i ae Motor Control JKtron Program Focus Camera Single Image Fig 1 Main Menu 46 Main Menu of the JKtron Program As
11. Focus Camera Single Image Motor Control JKtron Program Ch 0 will appear the control panel will become enabled and Processing Channel will be JExperimentinfe 1 selected from the pull down menu throughout the ponen Fig 6 Start or stop JKtron To end an experiment push the QUTT button located at the right side of the control panel above the tab menu Fig 6 You will then be given the choice to End Now or Resume The program will continue to run until End Novv is selected Advanced Operations of the JKtron 1 Switching control panel during experiment As shown in Fig 4 Parameter Settings and Experiment nfo can be viewed by changing the value of the pull down menu These values serve only as indicators and cannot be changed while the experiment Is running The exception is Channel Notes under Parameter Settings 49 The contents of the Processing Channel age will be selected and displayed each Processing Channel RU 7 ei Processing Channel _RU time a new image 1s shown Fig 7 Adjustments to the image and colonies Add Delete Colonies are made here and can be made at any time Adjust Channel Parameters throughout the experiment Resize Move Individual Colonies Rotate Shift All Colonies 2 PAUSE function The PAUSE button replaces the RUN button after the experiment has started Push the PAUSE button to remain on a particular Image or channel for an Snap Coloni
12. Upload plate samples into table s wells 4 Select JKtron Program tab on the tab menu 5 Choose Experiment Info from the pull dovvn menu Fig 3 47 Experiment Info New Experiment _ Manual Restart Select Folder JKtron Experiments Enter Experiment Number Set for time untl AUTOMATIC RESTART 15 m Fig 3 Experiment Info Note This pull down menu has 3 options and 1s located on the left of the control panel below the El y Experiment Info RUN button Fig 4 Parameter Settnga Processing Channel VAN RUN 6 Mark New Experiment If a new JKtron Manual Restart experiment is being started Fig 4 JKtron Program Tool Bar 7 Choose the folder you would like to save your experiment in Note Usually JKtron experiment data are saved to a folder named JKtron Experiments 8 Enter an experiment e g JK0001 JK0002 JK0003 9 Input an Automatic Restart time in minutes in case the program instead of power outage problem is hung up due to an unexpected reason e g 5 min 10 min 20 min etc 10 Choose Parameter Settings from the pull down menu Fig 4 11 Set parameters for each of the channels that are being tested Fig 5 including see Advanced Operations for more details Ch 1 ICh 0 Fig 5 Parameter Settings 1 Exposure time in seconds
13. agar plates 20 EE 21 ll 11710 b s 21 DREI AT 21 c ACQUISITION aaa 28 ins 0 OCO ii a A 28 ANS r 30 III FEASIBILITY OF LUCIFERASE ACTIVITY AS A BIOSENSOR OF ISOBUTYRALDEHYDE PRODUCTION IN CYANOBACTERIA 32 Preparatory Methods eeen aii 32 RES 34 Wa ON BL E 37 Future Prospects for Biofuel Production 37 A Future Application Kondotron to Screen for Fluorescence in Bacteria 38 Limitations of current fluorescence screening techniques 38 Measuring fluorescence in bacterial colontes 39 Appendix A KONDOTRON SOFTWARE SPEC ICS E 40 B USER MANUAL FOR OPERATION OF PROGRAM 46 Od GeO uu b m eee eas m nli 40 Operational Manta ia 40 REFERENCES EE 60 111 Figure z 22 z 2 4 20 2 6 l 3 2 25 2 A 3 4 A S LIST OF FIGURES Page Schematic depicting hardware connections of Kondotron 22 Photos depicting hardware connections of 40 22222 23 Flow chart depicting general software connections of new program 25 Flow chart depicting software connections of automated Kondotron program 26 Flow chart depicting image acquisition procedure
14. energy content than ethanol Atsumi et al have engineered E coli to produce isobutanol 1 butanol 2 methyl 1 butanol 3 methyl 1 butanol and 2 phenylethanol from glucose The general approach involved modification of the amino acid biosynthesis pathway to increase production of the 2 keto acid specific to the desired alcohol With regard to isobutanol production the acetolactate synthase enzyme alsS of Bacillus subtilis having a higher affinity for pyruvate than the corresponding E coli enzyme was overexpressed resulting in a 1 7 fold increase in isobutanol production Genes responsible for generation of by products that compete for pyruvate were deleted to further increase production The result was a strain of E coli capable of overproducing Isobutanol with a metabolic engineering approach applicable to the production of other alcohols and possibly other organisms Atsumi et al 2008 Using that same metabolic strategy C cellulolyticum was recently engineered to synthesize isobutanol directly from cellulosic material Strains were grown on agar plates containing either crystalline cellulose or cellobiose an Intermediate disaccharide product of cellulose Despite a spontaneous frameshift mutation in the alsS sequence resulting in a truncated protein the strain produced isobutanol at levels of 140 mg L on cellobiose and 400 mg L on cellulose a significant increase compared to the empty vector strain which reached just 17 mg L and 30 mg
15. in the separate windows will only be closed upon clicking the Close All Images button Troubleshooting 1 Hang up problem If an ongoing experiment 1s frozen or hung up due unexpected reasons the JKtron needs to be restarted manually Focus Camera Single Image Select Experiment Info tab Motor Control JKtron Program Mark Manual Restart box Experiment Info e Choose the folder your current data has been saved Select Folder e Open the JKtron experlment that needs to JKtron Experiments 11 be resumed Enter Experiment Number e Enter Automatic Restart time In minutes Set for time unti 1 AUTOMATIC RESTART e g 10 min Push RUN to start Fig 14 Manual Restart 2 Table turning problem If the turning table cannot be controlled by the motor controlling program the communication should be reset e Click Motor Control on the tab menu e Click Refresh to refresh the the motor s connection to the computer Fig 2 Note This should only be used when the movement function buttons do not result In motor movement 58 J K tron Exp User Date Project Protocol Temperature Light Startime Keys for special Channels 59 REFERENCES 2010 Energy Information Administration International Energy Outlook 2010 US Department of Energy Abualta B amp Withey J 2010 November 19 Genetic screening for bacterial mutants In liqu
16. industry and transportation sectors and is expected to remain the world s largest source of energy Energy Information Administration 2010 There exists therefore a tremendous need for the development and industrialization of energy dense biofuels to replace the petroleum based fuels currently in consumption Biofuels are defined as any fuel derived from biomass biological material from living or recentl y living organisms and they possess much potential as a substitute for gasoline and diesel fuels Meeting energy demands in a sustainable fashion will require the development of a large scale cost effective and carbon neutral energy system Lewis amp Nocera 2006 Bioethanol and biodiesel Liquid biofuels include ethanol and biodiesel Ethanol is drinking alcohol and is made from the fermentation of sugars while biodiesel 1s produced by the transesterification of lipids Demirbas 2009 Ethanol a two carbon alcohol is currently the primary biofuel in use with production exceeding fifteen billion gallons per year Fischer et al 2008 It serves largely as an additive in petroleum derived gasoline with its concentration varying greatly While ethanol has a higher octane rating and produces lower emissions its energy content is only two thirds that of gasoline per unit Efficient ethanol consumption requires a high compression engine that is not widely available Further existing methods of production are energy and cost intensive
17. involving several steps The first and most expensive step is procurement of simple sugars from the organic carbon source which is typically carried out by the addition of enzymes Various yeast strains then convert the sugars into ethanol through a process of fermentation distillation and dehydration Service 2010 Biodiesel refers to fuel consisting of long chain esters derived from organic oils orfats Its energy content exceeds that of ethanol and increases as hydrocarbon chain length increases Biodiesel is most often made by chemically reacting lipids with alcohols Common feedstocks include the oil of soy beans rapeseed sunflowers peanuts and palm trees while innovative research is examining the possibility of production by algae and other microorganisms Biodiesel is Europe s dominant renewable fuel Food crops Ethanol is manufactured predominantly from corn derived starch in the U S and sugarcane derived sucrose in Brazil but can also be produced from the polysaccharides of potato and rice plants Biodiesel 1s also manufactured chiefly from food crops with soybean oils being the most commonly used Despite high production numbers achieved there are several foreseeable problems with widespread employment of food crop based biofuels First there exist some basic concerns regarding the availability of food and water to the world s growing population As land is chosen for farming either food crops or ethanol producing cro
18. its wild type counterpart under constant light Xu et al 2000 However in 12 hour light dark cycles the wild type strain outgrows the inactivated strain Circadian gene expression is regulated by KaiA KaiB and KaiC proteins Some metabolic proteins such as the D1 protein of the photosystem II reaction center encoded by psbAl have been found to be circadian controlled Johnson amp Golden 1999 Liu et al 1995 It is unknown whether any of the genetic pathways controlling biofuel production are circadian controlled 14 Bioluminescence Bioluminescence refers to the production and emission of visible light in a living organism t is controlled by a chemical reaction that oxidizes luciferins a class of light producing organic substrates This conversion of chemical energy to radiant energy is direct and efficient meaning that very little heat is given off In the process Luminescence 1s also termed cold light Bacterial luciferin 1s a reduced flavin mononucleotide FMNH The luminescence reaction is catalyzed by luciferase and requires the oxidation of FMNH and a long chain aldehyde Hastings 1983 The result is emission of excess free energy in the form of a blue green light as represented by the following reaction FMNH RCHO O gt FMN RCOOH H20 light 490nm Long chain aldehydes C8 C 14 result in the most stable reactions Bioluminescence has been used as an iz vivo biosensor in cyanobacteria to ide
19. linear arrangement of thirty 20 mL vials each in a temperature controlled holder A phototube measured the luminescence of each sample moving from one position to the next under the control of custom software The automated system allowed for luminescence measurements to be taken over long periods of time thereby allowing for detection of certain luminescent rhythms Taylor et al 1982 An updated Taylortron apparatus showed that luciferase activity can accurately report circadian rhythms in cyanobacteria Gene expression activity was reported with bacterial luciferase uxAB fused to a promoter gene Period and phase changes were investigated by resetting the circadian clock with dark pulses thus resetting the luminescence pattern This provided evidence for the existence of circadian cycles in prokaryotes in liquid culture Kondo et al 1993 Circadian rhythm detection on agar plates The following two studies did not employ mechanical components but are significant in providing evidence of circadian rhythm detection via bioluminescent cyanobacteria imaged on agar plates 19 Subsequently luciferase was shown to be useful as a bioluminescence reporter for individual bacteria colonies Colonies grown on agar plates were found to display a rhythm similar to that of bacteria in liquid culture A cooled CCD camera 8 bit resolution took repeated images of a single agar plate Images were analyzed for luminescence intensity chang
20. plot scale automatically adjusts to colony intensity values and can be displayed with or without an additional plot gray of the plate s average colony Intensity over time Analysis Analysis software affords for additional calculations and manipulations The program provides calculations of the minimum maximum mean and integral luminescence for each colony over the course of the experiment Period phase and luminescence could be adjusted to account for all data or a desired portion of the acquired data Baseline calculations could also be adjusted in this manner A slideshow containing a plot and calculations of each colony is displayed with the abilities to pause and control slideshow speed Specific images can be saved with the displayed contrast and brightness settings and opened in other commonly used programs such as Microsoft Office Windows Live Photo Gallery and Paint Similarly a series of 28 JOSOI 918 SUOTIVINGIJUOD eJoureo pue 5 SI soaeuir Y A BULY p p x uooq SPY oui 5 0 IY vo VIP ISPUIL SYI A enunuoo yey doot e Jo sjsrsuoo 2301 2080 YI9H msodx v seys pue BIDUILI n SOINSIJUOS uonismboy ISP jenrul ure1304d p jgutolne v JO pts no pue ulu ujoq sbur ue Surmmboae u uA sinooo sdojs Jo sonos sim mrmp ooid uonismboe oSeur SUNIIdIP yeyo MO S e DANS as san dos e3eq a3eu peay y so pueH a3eu 3 li uado JAW WIS ejJ
21. produced by photosynthesis are directed toward H production Cyanobacteria are able to produce H gt through native hydrogenase native nitrogenase and an introduced hydrogenase However these enzymes are inactivated by O gt thereby limiting the efficient production of H Thus research has resorted to separating H gt production from photosynthesis through use of specialized cells with low concentrations and O gt depleting sulfur deprivation Metabolic engineering has increased H gt production through disruption of Calvin cycle flux Photobiological H2 production has managed to surpass rates of current crop based biofuels The opportunity remains for cyanobacterial production of higher energy fuels but it is possible that hydrogen gas production could also be optimized for use as an additional fuel source Angermayr et al 2009 McKinlay amp Harwood 2010 Isobutyraldehyde One such higher energy fuel of increasing interest 1s 1sobutanol Isobutyraldehyde IBA can be converted to isobutanol and other hydrocarbons with chemical catalysts Advantages of isobutyraldehyde isobutanol biofuel over ethanol include higher energy density higher octane number less corrosion to pipelines due to lower hygroscopicity ability of a substance to extract water molecules from the surrounding environment and an easier purification process due to its high vapor pressure Atsumi et al 2008 11 Stemming from their earlier work with E
22. shown in Fig 1 there are four categories of function 1 Motor Control for table turning control 2 JKtron Program for JKtron experiment setup 3 Focus Camera for camera s focus and zooming 4 Single Image for taking individual images Note On top of the tab menu shows total running time exposure counter seconds current temperature of the camera indicator for positioning turns green when a position Is reached and QUIT click 1t to stop running the program Basic Operations of the JKtron Program 1 Click Jktron program vi on desktop to open the program 2 Click Motor Control on the tab menu to move the table Fig 2 1 Choose desired direction Counterclockwise ys clockwise and speed Fast vs Slow of motor 2 Select Scan Step Home or Channel 0 11 to start the motor moving with the desired function 3 Click Stop Motor to stop the table from turning immediately Counterclockwise recton a Fast Speed Channel 0 STOP Motor Refresh Connection Fig 2 Motor Control Note Table must pass Home position before properly selecting a particular channel The position Indicator will light up once the table has reached the desired location and the motor has stopped Click Refresh to refresh the motor s connection to the computer This should only be used when the movement function buttons do not result in motor movement 3
23. 2009 Microorganisms involved in fungal decomposition of plants and termite digestion possess the innate ability to degrade lignocellulosic materials and convert the hydrolyzed products to ethanol Various enzymes are secreted and the genetic pathways encoding for these enzymes are being explored through genetic recombination Alper amp Stephanopoulos 2009 Yeast wild type S cerevisiae strains readily ferment hexose sugars vla glycolysis but contain no Innate pathway for pentose fermentation Recent metabolic engineering studies on S cerevisiae stralns expressing pathways for pentose fermentation have enabled rapid and efficient fermentation of xylose via a fungal xylose Isomerase pathway but require further optimization of the arabinose fermentation pathway van Maris et al 2006 Fernandes c Murray 2010 Cellulolytic anaerobic Clostridia strains are naturally able to produce cellulase hydrolyze cellulose and ferment sugars including xylose Yet these bacteria also naturally produce acetate and lactate reducing the efficiency of ethanol production Demain et al 2005 Metabolic engineering of Clostridia has proven difficult in comparison to that of non cellulolytic yeast and E coli strains Fischer et al 2008 C cellulolyticum however has been successfully engineered to increase ethanol production by 53 Guedon et al 2002 Microbes have also been metabolically engineered to make higher alcohols which possess greater
24. L respectively The WT engineered strain produced 364 mg L on cellobiose and 660 mg L on cellulose Difficulties relating to the gene expression system and enzymatic activity measurements were encountered with C cellulolyticum but such difficulties were not present with E coli Higashide et al 2011 Regardless heterotrophic microbes require an organic carbon compound source Whether the sugars come from starch cellulose simple glucose or some other source the carbon remains an expensive medium Feedstocks are the bulk of the cost in these biofuel applications comprising approximately 80 of the cost This cost includes producing harvesting storing and transporting the organic carbon source Babcock 2007 Dismukes et al 2008 Algae Photosynthetic capability 1s a promising target for biofuel generation as photoautotrophs do not require an organic medium The growth rate of algae 1s much faster than that of plants potentially allowing for more efficient fuel production However algae require a lot of water and atmospheric levels of CO aren t high enough to spur exponential growth Moreover the algal photosynthetic approach requires several intermediate steps for recycling CO into usable fuels and an extensive extraction process thereby resulting in high production costs McKinlay amp Harwood 2010 Sheehan 2009 Ethanol from aleae 1s possible by converting starch the storage component and cellulose the cell wall c
25. LUMINESCENCE ACTIVITY AS A BIOSENSOR OF ISOBUTYRALDEHYDE PRODUCTION INCYANOBACTERIA by Jill Mecklenborg Thesis Submitted to the Faculty of the Graduate School of Vanderbilt University In partial fulfillment of the requirements for the degree of MASTER OF SCIENCE In Biomedical Engineering August 2011 Nashville Tennessee Approved Carl H Johnson Robert L Galloway Jr TABLE OF CONTENTS Page ISIS TOF FIGURE EE EE iv Chapter I INTRODUCTION E I The Growing Importance of Bioluels 1 Bioc ihanolundbodicsel a 2 eege 3 I3 nocelIultoste ee E 4 Heterotrophie EEN nasa 6 2100 o ee 8 Cyanobacterial Biofuel Production 9 See EE 11 obutyaldeoliyde m a qa 11 Ad d nonalh sl enerey TUe issus 12 Optimization of Biofuel Producing Cyanobacteria 13 Manipulation of circadian pathways 14 O 15 A NOVE FIJ C WEE 16 II A HIGH THROUGHPUT LUMINESCENCE SCREENING SYSTEM 17 Expte ALO EE 17 Bioluminescence as a reporta 17 Hish throushput SM 18 System Development EDStOEy in 19 Automated luminescence measurements in liquid culture 19 Circadian rhythm detection on agar plates 2 19 Automated luminescence measurements on
26. Microbiol Biotechnol 5 495 509 Johnson C amp Golden S 1999 Circadian programs in cyanobacteria adaptiveness and mechanism Annu Rev of Microbiol 53 389 409 Klemm D Heublein B Fink H P amp Bohn A 2005 Cellulose Fascinating Biopolymer and Sustainable Raw Material ChemInform 36 36 Kondo T amp Ishiura M 1994 Spril Circadian Rhythms of Cyanobacteria Monitoring the Biological Journal of Bacteriology 176 12 1881 1885 Kondo T Strayer C A Kulkarnit R D Taylor W Golden S S Ishiura M et al 1993 June Circadian rhythms in prokaryotes Luciferase as a reporter of circadian gene expression in cyanobacteria Plant Biology 90 5672 5676 Kondo T Tsinoremas N F Golden S S Johnson C H Shinsuke K amp Ishiura M 1994 November 18 Circadian Clock Mutants of Cyanobacteria Science 266 1233 1236 Leach K Stroot J amp Lim D 2010 October 29 Same Day Detection of E coli O157 H7 Recovered from Spinach Using Electrochemiluminescent and Cytometric Bead Array Biosensors Appl Environ Microbiol Lewis N S amp Nocera D G 2006 October Powering the planet Chemical challenges in solar energy utilization PNAS 105 43 15729 15735 6l Lindberg P Park S amp Melis A 2010 Engineering a platform for photosynthetic isoprene production in cyanobacteria using Synechocystis as the model organism Metabolic Engineering 12 70 79
27. ON IN CYANOBACTERIA Gas chromatography and high performance liquid chromatography are the current methods most commonly used to quantify alcohols and aldehydes Such techniques are expensive time intensive and low throughput Therefore we have designed a method to use luminescence as a non invasive gauge of isobutyraldehyde CH3 2CHCHO production This is possible because the substrate necessary for the bacterial luciferase reaction is an aldehyde While optimal aldehyde chain length is between eight and fourteen carbons isobutyraldehyde with its two methyl groups on the carbonyl carbon 1s shown to be an acceptable substrate This bioluminescence reporter system will be used to select for mutants exhibiting enhanced aldehyde production The aldehydes can then be enzymatically converted to alcohols that can be used as fuel Preparatory Methods Two different cyanobacterial promoters were attached to luciferase psbAlp luxAB and trcp luxAB Individual cyanobacteria colonies were seeded onto agar in petri dishes The plates were exposed to different concentrations of isobutyraldehyde IBA vapor by means of a cap in the dish containing 0 2mL IBA dissolved in mineral oil 32 Results Initial experiments involved eight plates each containing cyanobacteria strains with the two aforementioned promoters Colonies were exposed to increasing concentrations of IBA for various exposure lengths It was found that as the concentration of IBA va
28. awes Wels s pueH 1 s u o E S A E uO 9e 1qns punossy9eg qQe IeAV GE 31815 J NYS uu 1 insodx4 SJ9 19UJBJ8d 2 19S ejeg 8 m S A eje uon s nb Jasay X anes 120 nisinboy uonisinbov ageuJ u21e4 93eu1 918114 spull 29 Process Image Save Data amp Settings Save Colony Coordinates to File Initiate Image Processing Determine Processing Ch amp Cycle u Load Ch Data x amp Settings E Load Image File Apply Threshold Apply Morphologies Filter Colonies by Size Save Colony Intensities to File Save Channel Parameters to File Obtain Bounding Colony Quantification Determine Intensity Rectangle Add or Delete Colonv No All Colonies Display Image with Properly Selected Colonies Selected Move or Resize Colony Display Colony Plot Figure 2 6 Flow chart depicting image processing procedure The newly acquired image 1s loaded and processing occurs according to parameters If colony selection displayed is not optimal parameters can be adjusted or colonies can be added deleted moved or resized 30 images can be saved as an Audio Video Interleaved AVI file The user is able to save the entire image or a selected portion 31 CHAPTER III FEASIBILITY OFLUCIFERASE ACTIVITY AS A BIOSENSOR OF ISOBUTYRALDEHYDE PRODUCTI
29. coli Atsumi et al engineered Synechococcus elongates PCC7942 to produce isobutanol directly from CO First the gene encoding for keto acid decarboxylase an enzyme that catalyzes the conversion of a keto acid precursor 2 ketoisovalerate to isobutanol was inserted and expressed under the control of the isopropyl B D thiogalactoside IPTG inducible promoter Ptrc Flux to 2 ketoisovalerate was increased when the alsS gene from Bacillus subtilis and the ilvC and ilvD genes from E coli were inserted These genes express enzymes involved in the conversion of pyruvate to ketoisovalerate Atsumi et al 2009 Rubisco catalyzes the CO fixation reaction in the Calvin cycle Overexpression of the rbcLS gene enhanced Rubisco enzyme activity in vitro and 1mproved isobutyraldehyde isobutanol production While the engineered strain showed growth retardation with high levels of isobutanol the isobutyraldehyde production strain produced constantly for nine days 1n the system The rate of production of IBA was higher than those reported for ethanol hydrogen or algal lipid production The coordinating increase in Rubisco activity and isobutyraldehyde production suggest that CO fixation 15 the rate limiting step Further optimization of CO fixation would likely improve IBA production Atsumi et al 2009 Additional high energy fuels The cyanobacteria Synechocystis was modified to produce isoprene an energy rich 5 carbon hydrocarbon used industrial
30. e Research 13 71 79 van Maris A Abbott D Bellissimi E van den Brink J Kuyper M Luttik M et al 2006 Nov Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae current status Antonie van Leeuwenhoek 90 4 391 418 Wijffels R H amp Barbosa M J 2010 August An Outlook on Microalgal Biofuels Science 329 796 799 Xu Y Mori T amp Johnson C 2000 Circadian clock protein expression in cyanobacteria rhythms and phase setting 19 3349 3357 Zhou J amp Li Y 2010 March Engineering cyanobacteria for fuels and chemicals production Protein Cell 1 3 207 210 63
31. ended product and development of conditions that would halt or cease production Therefore 1t 1s essential to screen a large library of mutants possessing the potential to produce the desired product Since the eventual goal 1s to produce large quantities of a biofuel for widespread use it is likely that the preeminent mutants will be those producing the greatest amounts of the intended biofuel throughout an extended period of time Bioluminescence as a reporter t is first necessary to find a means of quantifying the amount of biofuel produced Bacterial luciferase fused to a promoter sequence produces luminescent colonies that can be imaged and quantified Bioluminescence has been used to detect circadian rhythms in mutant colonies through analysis of images taken over a period of time Thereby luminescence serves as a real time reporter of circadian gene activity Bioluminescence could be used to detect the amount of biofuel production in a similar manner Long chain aldehydes are the substrate of a luciferase reaction producing a complimentary alcohol along with light emission These aldehydes and alcohols can be 17 used as high energy biofuels Of primary Interest 1s detection of isobutyraldehyde production Using a cooled CCD camera capable of sensitive light detection luminescent colonies can be detected and their luminescence quantified Bacterial luminescence on agar has been found to increase with known increasing concentrati
32. es over time Individual colonies were recognized as ovals by the custom software and luminescence data was taken from the selected regions of interest ROIs Mutants with varying period lengths were identified Kondo amp Ishiura 1994 Additional studies conducted around the same time showed transgenic Arabidopsis plants utilizing a firefly luciferase construct to be capable of reporting circadian patterns A video imaging system was used to visualize images of plant seedlings grown on an agar plate in low light conditions Luciferase bioluminescence was measured over time and mutants with varying period lengths were detected This system provided evidence that circadian rhythm luminescence could also be imaged from plant seedlings grown on agar plates Millar et al 1995 Automated luminescence measurements on agar plates This research led to the development of a high throughput apparatus that could screen for circadian rhythm mutations on multiple agar plates This Kondotron consisted of a stepper motor controlled turntable containing twelve channels for agar plates The apparatus employed a Macintosh based system with a custom driver interface Using this high throughput system cyanobacteria colonies were screened after 20 mutagenesis short as low as 16 hour and long up to 60 hour mutants were identified Kondo et al 1994 Current System Hardware Given significant advances in technology over the last thirty
33. es to New Position Undo Return to Colonies of Previous Apply Selection to Past 4 Future Ima Apply Selection to Future Images On extended period of time For example AAA pause running until completion of a Fig 7 Processing Channel manual colony selection Should the PAUSE button accidentally be clicked or the user have clicked the button and left the room the experiment will resume according to the pre set parameters after 30 minutes 3 Data Checking Click on a colony to display its Intensity plot selected colonies on that plate to be show Click on a new colony and its plot will replace the previous plot Click anywhere else on the screen to get the plot to disappear Right click the plot to indicate whether you would like the average intensity of all e Hold down the control key and click colonies to view up to five different colonies on one plot 4 Adjust appearance of image Appearance of image can be manually adjusted using Brightness amp Contrast Fig 8 Positive values will make the image brighter and show more contrast Negative values will make the image darker and show less contrast A more negative brightness value will make the background of the 1mage appear more black These settings do not affect intensity values of the image Settings will be saved and applied to future images of that channel 5 Adjust appearance of colonies Select display the colonies as Filled black or Outlined w
34. etabolism and habitat with some species thriving in salt water They would need to be grown in a land based photobioreactor system which is still expected to have a large area footprint However this could be placed on non farmable land thereby resolving the conflict between using land for biofuel or food crops Angermayr et al 2009 The major foreseeable problem with photoautotrophic growth 15 the rate limiting step of CO assimilation Zhou Li 2010 However this issue may be overcome as genome alteration of bacteria requires less extensive measures compared to that of plants Also with phototrophic microbes modifications to their nutrition and environmental surroundings can be easily made in an effort to maximize production Dismukeset al 2008 Ideally one cyanobacteria strain could be genetically engineered to produce high energy biofuels thereby simplifying the production process and eliminating the need for several steps to reach the end product Whatever the end product conversion to liquid fuel will require technologies simpler than those associated with breaking down the cellulose of biomass To improve biofuel production metabolic engineering strategies would need to re direct photosynthetic energy away from sugar production and toward the production of a biofuel Stephanopoulos 2007 10 Hydrogen Hydrogen gas itself can be used as fuel Hydrogen is an intrinsic end product of cyanbacterial fermentation as electrons
35. f images The images will be displayed continuously in STOP grayscale in a separate window Focus Click STOP to stop the camera Fig 12 Focus Camera e Motor Control JKtron Program Sin le Ima Focus Camera Single Image Click Single Image tab Set Exposure time in seconds Exposure Shutter Control lo Normal Background File to Subtract Choose a shutter control option a Normal vs Closed for background images Save Current Image As o Indicate what to save the image as by 1 entering a location and name in the space provided or 2 clicking B on the file folder icon to the right of the space and selecting a name and location via the dialog box e If you would like to subtract a background image 1 ie enter a location and name or 2 click on the associated file folder icon select via dialog box Click Acquire to start the exposure The time in the upper right corner will indicate the image exposure time e When the exposure is complete the most recent image will appear in color on the large area to the right of the control panel e appearance of this image can be adjusted using the Brightness amp Contrast box e The image will also appear in a separate window in grayscale When you take multiple images these also appear in separate windows allowing for quick comparison of different exposure times Fig 13 Single Image 57 The images
36. haride derived from glucose and fructose must first be broken down into simple sugars e g glucose and fructose This is usually carried out by the addition of enzymes which represent a major cost component of the overall process Ethanol derived from corn actually requires more energy input than it produces with one liter of 99 5 corn ethanol requiring 46 more fossil energy than the energy produced as ethanol Biodiesel from soybean plants requires 27 more energy Service 2010 Pimentel amp Patzek 2005 Lignocellulosic biomass In addition to edible polysaccharides sugarcane and corn also contain lignocellulosic biomass that can be made into biofuel Lignocellulose is the non edible fibrous or woody portion of plants It is the feedstock for the paper and textile industries but it can also be broken down into simple sugars and fermented to produce cellulosic ethanol Plants providing high yields of biomass per acre such as switchgrass and miscanthus can be grown at low cost with minimal agricultural effort In contrast to food crops these plants can be harvested multiple times each year and require significantly less water and fertilizer While production of ethanol from biomass would potentially alleviate some of the aforementioned problems new difficulties arise pertaining to the conversion of lignocellulosic material Into sugar monomers Somerville et al 2010 Lignocellulose is composed of cellulose and hemicellulose pol
37. he fatty acids The increased fatty acid production and secretion seen in mutants suggests that fatty acid secreting cyanobacteria may be a capable technology for renewable biofuel production Liu et al 2010 Optimization of Biofuel Producing Cyanobacteria It is important to note that cyanobacteria can complete more than one growing cycle in a single day with some species doubling in just a few hours of culture 13 Identification and manipulation of these metabolic pathways could increase their biofuel producing capacity Increasing knowledge of their physiology development of advanced technologies In photobioreactor design and manufacture of high energy products would improve the economic feasibility of the cyanobacterial production system Manipulation of circadian pathways In cyanobacteria as well as in the majority of organisms there exist genetically programmed daily cycles known as circadian rhythms These clocks consist of intricate mechanisms of chemical reactions responsible for regulating the timing of cellular events Circadian pathways control metabolic processes including cell division Mori amp Johnson 2001 By growing strains in competition with each other cellular fitness was determined to be maximal when phasing of circadian processes closely matched that of environmental light dark cycles Ouyang et al 1998 It was found that the S elongatus PCC7942 strain with an inactivated circadian clock outgrows
38. he system would involve a few additional components Fluorescent measurements would require an excitation system the first optical system to illuminate the colonies using a specific wavelength selected by an optical filter An emission system a second optical system would be needed to collect the emitted fluorescent light This light is then separated from the excitation light using a filter and the fluorescent signal is measured with a light detector such as a photomultiplier tube PMT This application would provide greater sensitivity as well as greater application range given the wide selection of fluorescent labels available today Luminescence reporter systems with their light emissions being the result of a chemical reaction instead of light excitation are simpler optically than their fluorescence counterparts as they only require a light detector and do not require a light source 39 APPENDIX A KONDOTRON SOFTWARE SPECIFICS This appendix consists of five figures depicting the LabView block diagrams used to code the newly developed program t 1s meant to be an extension of the Current System sectlon of Chapter 2 and as such clear and direct parallels exist 40 euim Aue pojrxo oq upo 0 8 DY Apuejsuoo s vuoureo ou PVPs JoyJOUe pue p pu PAs aq ue suondo noy Jo AUL IY WOI st doo o rqA e pezi enrur InJssooons 318
39. hite option to display colony number on image 50 Contrast U 25 0 0 10 2025 Brightness n 100 50 o 50 100 Apply Automatic Threshold Pixi t I I 20 40 60 80 100 120 140 160 20 Min Max Small Colonies Are I I 8 50 100 150 200 250 300 350 400 Min Max Fig 8 Adiust image 6 Zooming and panning Click the magnifying glass icon on the image tools menu to zoom In on the image Fig 9 Click on the image on the location at which you would like to zoom Repeat to zoom in further Note The image tools menu i1s at the bottom right of the image and consists of Zoom to Fit and icons of a magnifying glass an arrow a hand and an oval To move to different locations on the image use the image scrollbars or click the hand icon and drag Fig 9 Zooming Click Zoom to Fit to fit the entire image on the screen Adjust colonies selected by choosing different options on the colony selection menu Colony selection can be changed at any time throughout the experiment Note The colony selection menu 1s located on the Processing Channel page directly under the pull down menu Adjust the scrollbar at the right of the menu to show more 7 Add Delete Colonies This 1s the default setting and will be selected at the beginning of each image with Delete Colony and Add Colony unticked and the oval tool selected Fig 10 This option allows for the manual add
40. hough segments never overlap the resultant selected colonies may overlap slightly because all colontes are converted to ovals e Separation via Erosion separates selected pixels of the binary image that touch each other by narrow isthmuses 9 Resize Move ndividual Colonies This option allows the user to resize and or move a particular colony When the mouse is moved over a colony s boundaries a double headed arrow will appear Clicking allows for resizing of the colony by dragging the mouse When the mouse is on the center of a colony two double headed arrows in the shape of a cross Clicking allows the user to drag the colony to a new location New colony selections are automatically saved to be applied to future images 10 Rotate Shift All Colonies When selected a new control will appear in place of Add Colony and Delete Colony Rotate all colonies a specified number of degrees Increase values to rotate in the CW direction and decrease values to move in the CCW direction Shift all colonies a specified number of pixels Increase values to move colonies up and right Decrease values to move left and down New colony selections are automatically saved to be applied to future images Once the colonies have been set to a specific location all values are reset to indicate the newly selected position as zero 11 Snap Colonies to new Position This option is to be used when a plate has been moved for any reason Selec
41. id erowth media by fluorescence activated cell sorting J Microbiol Methods Alper H amp Stephanopoulos G 2009 October Engineering for biofuels exploiting innante microbial capacity or importing biosynthetic potential Nature Reviews 7 715 723 Andrew J Millar 1 A H 1995 February 19 Circadian Clock Mutants In Arabidopsis Idenitfied by Luciferase Science 267 1161 1163 Angermayr A S Hellingvverf K J Lindblad P amp Teixeira de Mattos M J 2009 Energy biotechnology with cyanobacteria Current Opinion in Biotechnology 20 257 263 Atsumi S Hanai T amp Liao J C 2008 January Non fermentative pathways for synthesis of branched chain higher alcohols as biofuels Nature 45 1 3 86 90 Atsumi S Higashide VV amp Liao J C 2009 December Direct photosythetic recycling of carbon dioxide to isobutyraldehyde Nature Biotechnology 27 12 1177 1180 Babcock B A 2007 Spring High Crop Prices Ethanol Mandates and the Public Good Do They Coexist Towa Ag Review 13 2 Demain A Newcomb M amp JH W 2005 Mar Cellulase clostridia and ethanol Microbiol Mol Biol Rev 69 1 124 154 Demirbas A 2009 Political economic and environmental impacts of biofuels A review Applied Energy 66 S108 S117 Dismukes C G Carrieri D Bennette N Ananyev G M amp Posewitz M C 2008 Acquatic phototrophs efficient alternatives to land based crops for bi
42. in acquiring images of plates at various lengths to determine optimal exposure time The most complex of the software features 1s the automation of a series of images Ktron Program which combines image acquisition turntable movement and image processing While an image 1s being acquired the previous image 1s displayed for processing The program can be paused at any time to allow for additional processing time Once running this program will continue to acquire and save images until it is stopped There now exists the ability to move individual colonies in the middle of an experiment so as to account for any drifting that might occur if the plate were to dry out or crack during the experiment Multiple colonies can also be moved as a single unit compensatory for accidental or purposeful movement of a plate 24 SWI Aut JE D JIX JY uk WEIOOIU YY PYJELWOJLLE UJ JO JUDPUIJUDPUL 15 OFLU PUL onuoo I QPJUIN SMO 918MIJOS MY WeISOId MIU JO suonoouuoo eIOUIS SUNIIdIP 11845 MOTA ez ANSIA SNILA uonnq LIND duon y N N WJOJI9A JIANNSIY JO ON pud o9JnjeJeduJa ePJQWED 195 a qequun sao 93eu 8u s uo43y UNY Main JO sinb y Eug sao VV ON uoneolunululo S A 9 qe1uJn 9 Usilqeqs4 LYVLS 25 A SUOYUEJ LLUIS SULSSIJOIA HUE UOLJISTLIDIG JOLLY jo uukuo JX9U v OJ SIAOW
43. ition and deletion of selected colonies 1 Deleting colonies When a colony is deleted it is deleted from all previous 57 images also Intensity values of the average plot will change _ Delete Colonies although the change may be slight Fig 10 Add Delete e Hover the mouse over or click the unwanted colony Push Delete on the keyboard e Tick Delete Colony Click the colony to be deleted This method is best for quick deletion of colonies 2 Adding colonies When a colony is added the new colony s coordinates are applied to all previous images to obtain intensity values for that colony The colony plot and average plot will indicate as such There are several ways to add colonies e First check that the oval icon on the image tools menu is selected It is impossible to add colonies if this 1s not selected e Draw an oval around the colony to be added starting from the center of the colony and expanding outwards 51 Push Insert on the keyboard Use the mouse to double click the newly dravvn oval Before drawing the oval tick Add Colony on the control panel Fig 9 Then draw the oval as above When the mouse 1s lifted the colony will be added Immedlately No adjustments can be made after the mouse is lifted but the colony can be deleted according to the above directions This method is best for quick adding of colonies and Is recommended for use only after the user has developed proficiency in adding col
44. l Production Isobutyraldehyde CH3 2CHCHO is a branched aldehyde consisting of just two methyl groups on the carbonyl carbon but affinity for FMNH increases with increasing chain length Ideal binding affinity 1s with a linear aldehyde of chain length between elght and fourteen carbons Expectations are that performance of this system will improve if long chain aldehydes alcohols or alkanes are the desired end product With increased affinity a greater percentage of the aldehyde will to bind to the FMNH resulting in improved sensitivity of luminescence reporting Further alcohols alkanes and aldehydes with longer chains provide more energy than their short chain counterparts The energy contained in the hydrocarbons of a long chain a highly branched chain or an aromatic ring 1s greater than that of short chain functional groups such as methyl and ethyl groups High energy content of such products 1s appealing for biofuel applications We plan to test additional alkanes alcohols and aldehydes using the established system 37 A Future Application Kondotron to Screen for Fluorescence In Bacteria Limitations of current fluorescence screening techniques Microplate readers are the primary screening technique for fluorescence in bacteria Plate readers employ a liquid media which 1s convenient In that most bacterial samples can be easily introduced into and grown in this type of medium High density microplates 384 or 1536 well mic
45. ly in synthetic rubber polymers The heterologous expression of plant isoprene synthase spS enabled photosynthetic isoprene generation Codon optimization of the DNA sequence conferred an advantage 12 In spS gene expression allowing for overexpression of the gene and increasing the yield of isoprene Lindberg et al 2010 Upon discovery of a new alkane synthesis pathway in cyanobacteria C13 C17 alkane alkene mixtures were produced in E coli Orf1593 and orf1594 genes were found to produce an acyl acyl reductase and an aldehyde decarbonylase implicating these as the genes responsible for the conversion of fatty acid 1ntermediates to alkanes and alkenes To test this hypothesis the alkane operon from cyanobacteria was expressed in E coli which 1s known not to produce alkanes The result was a strain of E coli that could produce alkanes proving that these genes and enzymes can be altered to control direct conversion to alkane biofuels Schirmeret al 2010 Additionally C10 C18 fatty acids were overproduced and secreted into medium by metabolic engineering of Synechocystis PCC 6803 First an acyl acyl carrier protein was introduced and found to increase fatty acid production This was followed by a series of genetic alterations that further enhanced increased fatty acid production More modifications were made by deleting the S layer from cell envelopes and weakening peptidoglycan layers This was successful in increasing secretion of t
46. ntify genes involved in circadian pathways luciferase gene luxA B is linked to the gene of interest and the luminescence pattern monitored Kondo et al 1993 Liu et al 1995 The alkJ luxAB with blaP promoter was recently found to express luminescence in the presence of aldehydes and alcohols To do so alkJ produces an alcohol dehydrogenase that converts C5 C12 linear primary alcohols to aldehydes Luminescence increased linearly with increased concentrations of IBA The long chain aldehydes produced can be converted to alcohols which possess a high energy density Minak Bernero et al 2004 15 A novel idea Genetically engineered cyanobacteria provide a promising system for both biofuel production and CO utilization It remains unknown as to which genetic pathways contribute to aldehyde production and to what extent The idea proposed here 1s to manipulate circadian pathways in an effort to optimize IBA production as this has not yet been studied The goal 1s to find the strain able to maximally produce isobutyraldehyde with a procedure applicable to the production of other biofuels The amount of product could be measured by luminescence levels 16 CHAPTER II A HIGH THROUGHPUT LUMINESCENCE SCREENING SYSTEM Explication The immediate objective 1s to find the cyanobacterial mutant or mutants that result in optimal biofuel production There are several factors to consider amount of intended product purity of int
47. ofuels Current Opinion in Biotechnology 19 235 240 Energy Information Administration 2010 International Energy Outlook 2010 US Department of Energy Fargione J Hill J Tilman D Polasky S amp Hawthorne P 2008 February Land Clearing and the Biofuel Carbon Debt Science 519 1235 1238 60 Fernandes S amp Murray P 2010 Nov Metabolic engineering for Improved microbial pentose fermentation Bioeng Bugs 1 6 424 428 Fischer C R Klein Marcushamer D amp Stephanopoulos G 2008 Selection and optimization of microbial hosts for biofuels production Metabolic Engineering 10 295 304 Gordon J amp Polle J 2007 Ultrahigh bioproductivity from aleae Applied Microbiology Technology 71 969 975 Guedon E Desvaux M amp Petitdemange H 2002 Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering Appl Environ Microbiol 68 53 58 Hastings J 1983 Biological diversity chemical mechanisms and the evolutionary origins of bioluminescent systems Journal of Molecular Evolution 19 5 309 521 Higashide W Li Y Yang Y amp Liao J C 2011 April Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose Applied and Environmental Microbiology 77 8 2727 2733 Jeffries T amp Jin Y 2004 Feb Metabolic engineering for improved fermentation of pentoses by yeasts Appl
48. olonies 3 More options for colony morphologies Other operations are often useful in the selection of small colonies but may be minimally useful in selection of medium or large colonies These operations are applied to the binary image after thresholding but before filtering the colonies by size The default is no additional operations Morphologies are applied first with a maximum of three applied in the order chosen These options are described below 53 Dilate increases the size of the selected area by increasing the area around the boundary This 1s in opposition to an Erosion which decreases the size of a selected area by decreasing the area around the boundary If two colonies have boundartes close in proximity Dilate may result in indentification of these two colonies as one colony similarly if one larger colony is identified as two smaller colonies applying the dilate function may result In identification of just one colony Applying an Open Close or Open amp Close function to an image before applying a Dilate function can work to smooth boundaries and separate colonies further before dilating them gt It seems especially useful when application of an automatic threshold results 1n the selection of colonies that are slightly smaller than the actual colonies Applying a dilation or multiple dilations in this case will likely increase the size of the colonies selected to produce the desired result
49. omponent Lipid production by algae seems a more efficient means given that algae lipid energy content greatly exceeds that of ethanol Thus algae hold the potential for producing both ethanol and biodiesel with lipids in aleae oll made into biodiesel and carbohydrates converted to ethanol Gordon amp Polle 2007 Stephens et al conducted industrial feasibility studies and sensitivity analyses to find that cost effective technologies have not yet been successfully integrated and optimized It was concluded that algal biomass may become an attractive option for biofuel production if metabolic engineering can boost productivity to offset the expensive extraction process Stephens et al 2010 Cyanobacterial Biofuel Production Phototrophic microbes hold much promise in the development of biofuels as the utility of photosynthesis bypasses the need for harvesting transporting and deconstructing plant derived biomass to provide simple sugars Unlike heterotrophic microbes their growth is inexpensive requiring only a salt medium Cyanobacteria also known as blue green algae are photoautotrophic prokaryotes capable of converting solar energy into chemical energy and fixing CO via photosynthesis Cyanobacteria are more efficient solar energy converters than plants thriving across a greater range of photon intensities while exhibiting greater light capture efficiency due to the ability to capture light all year They exhibit diversity in m
50. onies g Adjust Channel Parameters Apply Automatic Threshold Pi Choose this option to adjust the parameters for 1 1 I I i 1 20 40 60 80 100 120 140 160 automatic selection of colonies The program s automatically saves parameters selection and Small Colonies hee applies new colony selections to all previous Images of that channel before moving to the next 8 50 100 150 200 250 300 350 a vo Min ax image Figs 5 amp 11 Additional Processing Options 1 Threshold Image Morphologies Adjust Threshold via pull down menu and Separate Colonies via Watershed Transform sliders It is important to know that Threshold 1s the first parameter to be applied Fig 11 Adjust Channel Parameters when processing the image Here are some tips Check intensity levels of the image to get an idea of what values to use when applying a manual threshold The resultant image after thresholding 15 binary but that binary image will never be shown on the screen Pixel values between the Min and Max values are 1 selected pixels while pixel values outside Min and Max are O unselected pixels The Min value indicates that every pixel in the image above that value will be selected and considered as part of a colony Values below Min will not be considered part of a colony The Max value indicates that every pixel below that value and above Min will be selected and considered
51. ons of isobutyraldehyde vapor Thus in comparing mutant luminescence levels of biofuel producing cyanobacteria it would be possible to determine the amount of isobutyraldehyde produced e g the amount of IBA in each dish Those mutants with the highest luminescence values over time will be those optimal for isobutyraldehyde fuel production High throughput screening High throughput screening HTS allows a researcher to rapidly identify active genetic mutations modulating a particular bimolecular pathway HTS provides continuous real time data collection It is especially useful for screening a large library of genetic mutations Automation is a critical operative element of an effective and well designed high throughput apparatus Such a device would make use of motorized mechanical components a means of signal detection and a data processing system with functionality controlled by automated software Thereby an HTS system can simultaneously incubate and analyze numerous plates allowing for the parallel screening of thousands of mutant colonies 18 System Development Automated luminescence measurements in liquid culture Bioluminescence measurements were first automated using the Apple II microcomputer in the early 1980s with the advent of the Taylortron Fluorescent lamps provided the illumination necessary for cyanobacteria to undergo photosynthesis in a liquid culture environment This original apparatus consisted of a
52. osic ethanol research efforts Service 2010 Jeffries amp Jin 2004 Cellulosic ethanol has the advantaee of abundant and diverse raw material including the woody portions of food crops potentially relieving some of the land use concerns Nevertheless current sugar procurement and fermentation techniques are cost Intensive with only 40 of the blomass energy content converted to ethanol Service 2010 For cellulosic biofuel production to be economically viable heterotrophic microbes must be engineered to increase the rate and yield of both cellulose conversion and suear fermentat on Fischer et al 2008 Heterotrophic microbes Heterotrophic microbes are being studied as a means of alternative fuel production due to the ease of expanding their use to industrial scale bioprocessing New metabolic pathways that control cellulose degradation and biofuel production are being discovered and manipulated in an effort to yield the most efficient production of biofuel from cellulose Using recombinant technology heterotrophic microbes could potentially be engineered to perform direct conversion of lignocellulosic material to biofuel Thus 1t may be feasible for a single microorganism to do the job of what would previously have required multiple cultures Producing biofuels directly from cellulose known as consolidated bioprocessing 1s believed to reduce costs substantially compared a multiple step approach Alper amp Stephanopoulos
53. part of a colony while every value above will not This value should only be lowered in the event that the user notes some very high intensity levels that are not part of the colonies 52 Automatic Threshold accomodates for varying background intenisty within an Image No adjustments to Min and Max can be made Manual Threshold Dim provides the user with a truncated slide allowing for greater accuracy in threshold value selection at lower levels Manual Threshold Bright provides a scale with a greater range of values allowing the user to choose higher threshold values with the slider 2 Colony Size Adjust the Colony Size filter via pull down menu and sliders Colony Size is the last parameter applied to the Image Min 1s the smallest value in pixels that a colony can be Decreasing allows for smaller colonies sometimes selecting noisy pixels as colonies Increasing eliminates smaller colonies but a value too high can eliminate small colonies Max is the largest value that a colony can be Decreasing eliminates any colonies larger than the value small Colonies applies a unique filter to the image suited for selection of small colonies The scale 1s truncated to allow for accurate selection of smaller Min values Medium Colonies provides a more expanded slide scale ideal for selection of medium colonies Large Colonies provides a very expanded scale necessary only when selecting larger c
54. por increases luminescence activity also increases Cyanobacteria with the trc promoter initially exhibit greater luminescence activity than those with psbAl promoter trc cyanobacteria exhibit some detectable luminescence at 0 IBA while psbAl do not psbAI cyanobacteria increase in intensity over several days to reach intensity levels comparable to trc cyanobacteria but trc cyanobacteria do not appear to increase in intensity significantly throughout the same time course 33 3 d n EE D SE SEE a 22 e PT S Ed b E H 7 LF a Figure 3 1 15 minute exposures of trcp uxAB at eight different IBA concentrations Plates contain 0 0 01 0 1 0 5 1 5 10 and 25 IBA Mention that colony sizes were equivalent 34 q msodx nurur e pue q msodx nurur z e o juo eA mbo s dbuu p o Iduioo 4 y s msodx nurtu A SS nS ddI Jo SOLIDS V wl 256 UM soruo oo PIIJIBQOUR S YY XN d944 ee IIIA 35 5 po rduio g y s msodx lnurur q msodx ojnurur c e pue qq msodx ojnurur z e o ypAmb JAISSIDINS JO SITIOS V VAI YIM soruo oo erojoeqoue Ao qyxnj yqsd cc SANSIH 36 CHAPTER IV CONCLUSION Summary of Experimental Results This high throughput luminescence reporter system allows for efficient screening and monitoring of isobutyraldehyde producing mutants Future Prospects for Biofue
55. ps food supply could become limited and prices inflated The obvious solution is to extend farming to meet the world s food needs but extensive farming of these crops would worsen water pollution and cause localized water shortages The current corn farming process requires a great deal of water fertilizer and pesticides Corn ethanol and soybean biodiesel irrigation require two to three orders of magnitude more water than the oil extraction and refinement process Water usage would be reduced if seed companies are successful in engineering drought tolerant corn strains that would maintain their yields through extended dry spells Service 2009 Second there are concerns that the process of clearing sizeable portions of land for farming would result in significant carbon emissions Although biofuel feedstocks utilize CO the clearing of carbon rich habitats for farmland can actually increase greenhouse gases Searchinger et al 2008 As soil and plant biomass store large amounts of terrestrial carbon the burning and decomposition of natural habitats will result in significant CO release into the atmosphere Fargione et al 2008 Sheehan 2009 Existing methods of production are cost and energy intensive There 1s the Obvious cost of farming the food crops but fermentation and transesterification are costly procedures also In ethanol production the starch comprised of many glucose units joined by glycosidic bonds and sucrose a disacc
56. roplates are typically used for screening applications Bacterial colonies however do not form in a liquid suspension and a plate reader Is unable to measure the fluorescence of bacterial colonies on circular agar plates Agar 1s the most convenient way to grow bacteria because 1t does not require the laborious manual transfer of bacterial cultures into each well of a microplate Plating bacteria onto agar plates 1s advantageous for screening purposes because bacteria grow into single colonies each a clone of a single bacterium Fluorescent probes are commonly used in applications today Fluorescence activated cell sorting has been used to isolate bacteria grown in liquid media that have undergone mutations thereby permitting analysis of an entire bacterial population in real time Abuaita amp Withey 2010 Fluorescence based cytometric beads allow for the desired gene expression profile to be rapidly detected In liquid media but this methodology is not high throughput Leach Stroot amp Lim 2010 Measuring fluorescence in bacterial colonies Use of the Kondotron to measure fluorescence would allow bacteria to be grown on plates By using short wavelength LEDs emitting blue excitation light and putting an interference filter for green light emission on the current camera setup it 38 may be possible to establish a high throughput screening method for bacterial luorescence The application of fluorescence detection to t
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58. such colony adjustment Selecting Add Delete Colonies following such colony adjustment will result in the automatic application of this function Should the user want the new colony coordinates to be applied to previous images after one of these functions it 1s necessary to select Apply Colony Selection to Past amp Future Images 13 Apply Colony Selection to Past amp Future Images Selection will apply current colony coordinates to all past and future images thereby updating the intensity values associated with previous cycles When performed at a later cycle this function may take several seconds to complete This 1s the default option when Adjust Parameters 1s selected It 1s not necessary to select this option after adjusting parameters Selecting Add Delete Colonies following parameter adjustment will result in the automatic application of this function 14 Undo Return to Colonies of Previous Cycle Selection of this option will delete all adjustments made during the current cycle and return to the selected colonies of the previous cycle Each colony s intensity values will be returned to the values previously determined and the colony plots will indicate as such 56 Focus Camera Motor Control Tun Program Focus Camera 1 Single Image Exposure Click Focus Camera tab 10 00 Set Exposure time in seconds Adjust height of camera to focus Focus Camera Click Focus to start succession o
59. ting this option will result in the immediate movement of colonies to a new location This function moves all colonies together keeping them at approximately the same relative positions Use Resize Move Individual Colonies to further adjust the location of selected colonies If this option is selected on an image with very low contrast it is unlikely that the program be able to find the new location of colonies Scroll the mouse over the image to compare the intensity level of the background to that of the colonies If these values are only a few pixels different then this function may not produce the desired result 55 It is recommended to use the Rotate Shift All Colonies and Resize Move Individual Colonies functions to obtain the desired colony selection If selection of this function should produce an undesired result simply choose Undo Return to Colontes of Previous Cycle New colony selections are automatically saved to be applied to future images Special Attention Please be very careful when use Rotate Shift All Colonies and Snap Colonies to new Position 12 Apply Colony Selection to Future Images Selection will apply current colony coordinates to all future Images thereby keeping all past intensity values the same but using new colony coordinates to determine future Intensity values This is the default for Resize Move Rotate Shift and Snap It is not necessary to select this option after
60. ubtracts this image from all subsequent images in that cycle saving each image with the background subtracted Image Processing The image is opened and processed using LabView s Vision Assistant package Basic functions consist of brightness contrast adjustments colony display options and image zooming The brightness and contrast of an 1mage can be adjusted to allow for improved colony selection while the colonies can be selected as either a white outline or a filled black oval with the option to display colony numbers Selection of colonies is improved due to use of new image analysis techniques including various thresholding and morphological operations Thresholding can be automatic considers local pixel values or manual the user sets the threshold to a 27 particular value and all pixels above that value are selected A series of up to three morphological transformations can be applied to the thresholded Image including Dilate Open and Close The colony particles can be separated further by applying a Watershed Transform or simple erosion at the particle edges Finally the colonies are filtered by size eliminating any colony selections above or below the user set value When the user clicks on a colony a plot is displayed depicting that colony s intensity over the time course of the experiment Up to five colonies can be selected for comparison each displayed on the same image using different colors The
61. years the current system employs a commercially available stepper motor and driver a micro machined turntable and a modern PC computer The system features a turntable with channels for twelve individual petri dishes or plates Mechanical aspects are similar to the original system but the addition of a commercially available motor driver interface EMP400 Oriental Motors allows for all table functions to be handled by the computer as opposed to the manually switching of a physical control box The EMP400 controller is connected to the computer through a serial port The current system employs the PIXIS 512 camera Princeton Instruments which is a CCD camera water cooled to 70 C This camera has 16 bit resolution as opposed to the previously used 8 bit resolution The larger dynamic range provides an improved ability to distinguish quantitative differences in colony luminescence Software capabilities The software 1s PC based and run via LabView National Instruments providing the user with an efficient operative interface All turntable movement is now controlled with the software program with options for controlling the table independently of the 21 uonounjJ eJoureo s onuoo ose 1ojnduioo ou uoneorunururoo Jojnduroo 10 SurAo e 13 OMUOD dq ou YUM T ATIp pue 1olouu Ioddojs e Aq p l onuoo SI o qejuun MY Jo uorensrpuoo o1e paeu Surordop ncur q s VZ IANSIH ISAN 9SPUJ S 1o o3u07 44
62. ysaccharides tightly bound to lignin It thereby forms the structural framework of plant cell walls providing strength and rigidity to the stems stalks and trunks of plants Cellulose the most abundant of the three entails thousands of B D glucose units arranged in a linear chain making it tough and resistant to hydrolysis Hemicellulose consists primarily of pentoses the most abundant being xylose but also contains some hexoses and uronic acids It has a highly branched arrangement and contains fewer sugar units rendering it unstructured and weak in comparison to cellulose Lignin an amorphous polymer comprised of phenylpropanoid compounds crosslinks with cellulose and hemicellulose fibers via ether and ester bonds Klemm et al 2005 To extract the fermentable sugars the celluloses and hemicelluloses must first be disconnected from the lignin and then hydrolyzed into simple monosaccharides This process involves a costly combination of acid and cellulolytic enzyme treatments Due to 1ts robust structure obtaining lignocellulosic sugar monomers 1s more difficult and less effective than obtaining starch and sucrose monomers Yet another challenge involves the high percentage of pentose sugars in the hemicellulose fibers While the hexose sugars of cellulose starch and sucrose are fermented by typical yeast strains pentose sugars remain difficult to ferment Engineering yeast strains to metabolize pentose sugars 1s a leading area of cellul

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