Team:Nevada/Project

Introduction

Our project aims to overcome problems in modern oil reserve limitations by utilizing E. coli for the production of biodiesel (C-12 fatty acids) and bioethanol. In the past there have been a number of examples of biofuel production in E. coli, however 30-40% of production cost is based on media costs(Galbe, Sassner, Wingren and Zacchi, 2007). Our project will surmount these high production costs by engineering the cyanobacteria, Synechocystis PCC 6803, to secrete large quantities of glucose that will feed our biofuel-producing E. coli. In addition to cutting costs, Cyanobacteria and E. coli will be co-cultivated in an apparatus that allows for the mutual transfer of carbon to produce biofuels. This consumption of atmospheric carbon dioxide through photosynthesis provides an energy source that is beneficial to the environment. Not only will this project provide an efficient and environmentally friendly means for producing biofuels without the need for a carbon source, but the use of novel bacteria also frees up land and water reserves for food crops. The idea that two different bacterial species can be developed into a cooperative system also holds further industrial implications for medicine production and other endeavors.

Approach

E. coli

Fatty Acid Production

Cloning Stratagy
Bay Laurel thioesterase (BTE) is a gene that is naturally found in the bay leaves (Umbellularia californica) used in cooking. The BTE produces medium chain 12-carbon and 14-carbon fatty acids (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). The Nevada team changed the codon sequence for BTE based on the publication by Welch et. al. “Design Parameters to Control Synthetic Gene Expression in Escherichia coli” by changing the codons that are more common in plants to codons more common in E. coli without changing the polypeptide sequence. The synthesized gene also contained an E. coli ribosome binding sequence, a His tag sequence on the 3’ end of the coding sequence and an E. coli double terminator sequence (BBa_B0014) creating the Nevada thioesterase intermediate part BBa_K558003.

The constitutive promoter sigma 70 was used from the Registry of Standard Biological Parts (BBa_J23101) and was classically cloned in front of the synthesized Bay Laurel thioesterase gene to create the Nevada BTE generator part BBa_K558007. Many tests were used to determine the expression of the Bay Laurel thioesterase including SDS-PAGE, Western blot, colorimetric assay and gas chromatography. The one assay that was most successful for measuring free fatty acid content in the growth media was purchased through Bioassay Systems (EnzyChrom™ Free Fatty Acid Assay Kit www.bioassaysys.com). T7 Express Iq E. coli with the BTE generator produced up to 125 uM free fatty acid compared to control line which produced 50 uM fatty acid.

Ethanol Production

Pyruvate Decarboxylase and Alcohol Dehydrogenase Cloning Stratagy

The Nevada team designed and synthesized an operon without a promoter (intermediate part) containing pyruvate decarboxylase and alcohol dehydrogenase coding regions along with a standard E. coli ribosome binding site and double terminator. The pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) coding region sequences were based on the Zymomonas mobilis sequences (Ingram et. al. 1987) and work conducted by UNIPV-Pavia (iGEM 2009 Part K173016; K173017) and Utah State (iGEM 2009 M11041; M11042). The goal was to express this pdc/adh operon under the control of the constitutive promoter sigma70 (iGEM part J23101), or a glucose insensitive inducible promoter, trc (see details below) and test for expression of functional genes.

The sigma 70-pdc/adh generator (k558001) was assembled using classic cloning techniques. It was then transformed into two E. coli competent cell lines, NEB 10-Beta (New England BioLabs) and T7 Express Iq (New England BioLabs). Colonies were screened through restriction digest analysis, DNA sequencing and tested for enzymatic activity.

Pyruvate Decarboxylase Activity

Pyruvate decarboxylase activity was assayed using aldehyde indicator plates (Conway et. al. 1987) to test for a Schiff Base reaction to detect the presence of acetaldehyde, a substrate for ethanol production. The figure below shows pink colonies after an overnight incubation on Schiff base plates. The streaked colony on the top of the plate is a negative control cell line (white-light pink). The streaked colony on the right are a typical example (pink color) of sigma 70-pdc/adh generator in the NEB 10-Beta cells. The cells on the left (dark pink) and represent what is typically seen for the sigma 70-pdc/adh generator expressed in T7 Express Iq cell lines. From the results shown here and other experiments, we have concluded that the NEB T7 Express Iq cells are a better choice for expression of the 70-pdc/adh generator.

Alcohol Dehydrogenase Activity

Ethanol production was tested using EnzyChrom Ethanol Assay Kit (BioAssay Systems). The figure below shows percent ethanol production in T7 Express Iq cells (NEB) in 2% versus 10% glucose medium over 24 and 48 hours. The sample grown in 2% Glucose over 24 hours yielded 0.02% ethanol, and the sample grown in 10% Glucose over 48 hours yielded 0.018% ethanol. From the results shown here and other experiments, we have concluded that our modified E. coli is in fact producing ethanol. Furthermore, we have also concluded that glucose addition to the growth medium does have an effect on cell growth. It appears that these cells can only tolerate glucose additions up to 2.0% as the sample with a 10.0% glucose addition produced less ethanol.

E. coli Results

Cyanobacteria

The heart of the Team Nevada project is anchored in sustainability. As global warming continues to alter our planet, predictability has faded into little more than a nostalgic term used to describe a way of life that no longer exists. If life is going to continue on this planet, its success will depend upon the ability of its inhabitants to live harmoniously with their environment. Additionally, there will come a day when fossil fuels will no longer be available to humans for easy pickup and delivery and, on that day, the perpetuation of humanity will only be possible if an alternative to the quick-and-easy is developed. We, the University of Nevada team, believe that part of the solution to these problems lay in improving upon the natural sequestering of carbon dioxide from the atmosphere by photosynthesis through genetic engineering. The first criterion we established in searching for a photosynthetic organism to study was that it must have a rapid growth cycle. Immediately, green plants were eliminated as contenders and photosynthetic bacteria came to the forefront. Among the photosynthetic bacteria, one organism held the most promise: the cyanobacterium, Synechocystis PCC 6803. This bacterium has been studied extensively since its isolation decades ago and, today, its entire genome has been sequenced and is readily available through the internet database, “Cyanobase”. In addition to the abundant research surrounding Synechocystis and its rapid growth cycle, the organism can be easily transformed with exogenous DNA by natural recombination, making it an ideal choice for this competition. Above all, using bacteria to fix atmospheric carbon means that we do not have to dedicate agricultural land to the production of energy for our cars, it can remain untouched, as it should, to provide energy for our bodies and our minds.

The goal of this project is to engineer the cyanobacterium strain Synechocystis PCC6803 to produce and secrete hexose sugars that can be utilized by a biofuel producing E. coli strain. To achieve this goal the Cyanobacteria team has been assigned following two tasks
1) Engineer Synechocystis to overproduce the hexose
2) Engineer Synechocystis to secrete hexose sugar to the medium

Engineer Synechocystis to overproduce the hexose sugars

Cyanobacteria use photosynthesis to provide energy and carbon skeletons for anabolic processes. During the day excess fixed carbon can be converted to the polysaccharide, glycogen and stored for later use. To engineer Synechocystis to overproduce hexose sugars, we will divert carbon away from the glycogen biosynthetic pathway and towards hexose sugar production. To achieve this goal we will create a null mutation in the gene encoding ADP glucose pyrophosphorylase (AGP). AGP specifically converts glucose to ADP-glucose which is the monomeric precursor to glycogen. Synechocystis AGP knockout mutants have been reported to no longer produce glycogen, but instead accumulate high levels of sucrose (Miao et al (2003) FEMS Letters 218: 71-77). Fortunately, sucrose can easily be converted to the hexose sugars, glucose and fructose, in a reaction catalyzed by the enzyme invertase (INV). Therefore, we will introduce and overexpress the INV gene in the AGP mutant background.

Construct Design

We will simultaneously create the AGP mutant and the INV overexpressing line by inserting the INV expression construct into the coding region of the Synechocystis AGP gene. This will be accomplished by creating an AGP gene replacement construct in which the INV overexpression gene cassette and a kanamycin resistance gene cassette are inserted into the coding region of a subcloned AGP gene. This dysfunctional AGP construct will then be transformed into Synechocystis and through homologous recombination, replace the native AGP gene.

Construct Components

ADP Glucose Pyrophosphorylase (AGP) Gene: The slr1176 open reading frame was PCR amplified from total Synechocystis PCC6803 genomic DNA and subcloned into pSB1a3. Forward and reverse primers were designed with 5’ extensions containing iGEM prefix and suffix sequences.

Invertase (INV) Gene+double terminator: The INV gene was synthesized based on the sequence of a Zymonomas mobilis invertase gene described by Neiderholtmeyer et al. (Applied and Environmental Microbiology (2010) 76: 3462) which was codon optimized for expression in cyanobacteria. The gene was also synthesized with a double transcriptional terminator designed from sequences described for iGEM part BBa_B0015. The synthetic gene was subsequently subcloned into pSB1C and has been designated BBa_K558006.

petBD promoter+RBS: The strong light inducible petBD promoter will be used to drive the expression of invertase gene in Synechocystis. The petBD+RBS (BBa_K390015) was generously provided to us by the Utah State iGEM team.

Kanamyacin resistance cassette (KanR): The kanamycin resistance cassette was PCR amplified from pUC4K (Taylor and Rose (1988) NAR 16:358). This cassette includes the neomycin phosphotransferase 2 gene with its own constitutive promoter and transcriptional terminator. This cassette is frequently used to select for positive transformants in Synechocystis.

Gibson Assembly of Components:

Primer Design: Forward and reverse primers for each DNA part were designed with 20 base pair overlapping sequences with the upstream and downstream flanking segments. This will create 40 base pair overlaps between neighboring parts in the construct.

PCR: Inv, KnR, and petBD+RBS were amplified from BBa_K558006(Nevada), pUC4K, and K390015(Utah State) respectively under standard PCR conditions. AGP in pSB1A3 will be amplified to include the vector. This will be used as the backbone for Gibson Assembly.

Assembly of Parts: The above parts will be assembled into the following construct

Engineer Synechocystis to secrete hexose sugar to the medium

Synechocystis lacks the ability to secrete hexose sugars. Therefore it was necessary to introduce a sugar transporter through genetic engineering. Previous studies (Nedierholtmeyer et al (2010) Applied and Environmental Microbiology (2010) 76: 3462) showed that by expressing the Zymonomas mobilis glucose facilitative transporter (GLF) gene in Synechococcus, glucose and fructose were detected in the culture medium at levels as high as 30 M and 150 M respectively. Based on these results we decided to express GLF in Synechocystis. Our hope is that by expressing GLF in the AGP KO/INV overexpressing line, we will be able to produce enough glucose to the surrounding medium to support the E. coli biofuel production.

Approach

To integrate the GLF gene into the Synechocystis genome, we have decided to use an alternative insertion site. In this case we will insert the GLF overexpression gene cassette along with a chloramphenicol resistance cassette (CamR) into the coding region of the thiamin monophosphate pyrophosphorylase (ThiE) gene. The rationale for creating a ThiE knockout mutant is that it will lead to the creation of an auxotophic mutant that will only survive when the grown in the presence of thiamin (Vitamin B1). Therefore, the transgenic Synechocystis will not be able to survive outside laboratory and the chances of environmental contamination will be decreased. We will simultaneously create the ThiE mutant and the GLF overexpressing line by creating an ThiE gene replacement construct in which the GLF overexpression gene cassette and a chloramphenicol resistance gene cassette are inserted into the coding region of a subcloned ThiE gene. This dysfunctional ThiE construct will then be transformed into Synechocystis and through homologous recombination, replace the native ThiE gene.

Construct Design

Gene Construct Components

ThiE Gene: The sll0635 open reading frame was PCR amplified from total Synechocystis PCC6803 genomic DNA and subcloned into pSB1a3. Forward and reverse primers were designed with 5’ extensions containing iGEM prefix and suffix sequences.

Glucose facilitative transporter (GLF) Gene+double terminator: The GLF gene was synthesized based on the sequence of a Zymonomas mobilis invertase gene described by Neiderholtmeyer et al. (Applied and Environmental Microbiology (2010) 76: 3462) which was codon optimized for expression in cyanobacteria. The gene was also synthesized with a double transcriptional terminator designed from sequences described for iGEM part BBa_B0005.

petBD promoter+RBS: The strong light inducible petBD promoter will be used to drive the expression of invertase gene in Synechocystis. The petBD+RBS (BBa_K390015) was generously provided to us by the Utah State iGEM team.

Chloramphenicol resistance cassette (CamR): The chloramphenicol resistance cassette was PCR amplified from pSB1C3. This cassette includes the chloramphenicol acetyltransferase gene with its own constitutive promoter and transcriptional terminator. This cassette is frequently used to select for positive transformants in Synechocystis

Approach

To integrate the GLF gene into the Synechocystis genome, we have decided to use an alternative insertion site. In this case we will insert the GLF overexpression gene cassette along with a chloramphenicol resistance cassette (CamR) into the coding region of the thiamin monophosphate pyrophosphorylase (ThiE) gene. The rationale for creating a ThiE knockout mutant is that it will lead to the creation of an auxotophic mutant that will only survive when the grown in the presence of thiamin (Vitamin B1). Therefore, the transgenic Synechocystis will not be able to survive outside laboratory and the chances of environmental contamination will be decreased. We will simultaneously create the ThiE mutant and the GLF overexpressing line by creating an ThiE gene replacement construct in which the GLF overexpression gene cassette and a chloramphenicol resistance gene cassette are inserted into the coding region of a subcloned ThiE gene. This dysfunctional ThiE construct will then be transformed into Synechocystis and through homologous recombination, replace the native ThiE gene.

Gene Construct Components

ThiE Gene: The slr1176 open reading frame was PCR amplified from total Synechocystis PCC6803 genomic DNA and subcloned into pSB1a3. Forward and reverse primers were designed with 5’ extensions containing iGEM prefix and suffix sequences.

Glucose facilitative transporter (GLF) Gene+double terminator:""" The GLF gene was synthesized based on the sequence of a Zymonomas mobilis invertase gene described by Neiderholtmeyer et al. (Applied and Environmental Microbiology (2010) 76: 3462) which was codon optimized for expression in cyanobacteria. The gene was also synthesized with a double transcriptional terminator designed from sequences described for iGEM part BBa_K558006.

petBD promoter+RBS: The strong light inducible petBD promoter will be used to drive the expression of invertase gene in Synechocystis. The petBD+RBS (BBa_K390015) was generously provided to us by the Utah State iGEM team.

Chloramphenicol resistance cassette (CamR): The chloramphenicol resistance cassette was PCR amplified from pSB1C3. This cassette includes the chloramphenicol acetyltransferase gene with its own constitutive promoter and transcriptional terminator. This cassette is frequently used to select for positive transformants in Synechocystis.

Media Development

Assay Development

Quantification of Glucose/Fructose secretion Secretion will be tested using direct samples of Synechocystis media and a D-Fructose/D-Glucose assay. Assay Description: Invertase enzyme will be directly added to sample media to split sucrose into D-glucose and D-fructose, which are then added to a Hexokinase/Glucose-6-phosphate DeH assay mix, which will produce one NADH molecule for every one glucose molecule added. NADH can be measure on the spectrophotometer at 340 nm and can be quantitated using Beer’s law and the NADH extinction coefficient. Because assay is glucose specific, the first reading will quantitate glucose present, then a Phosphoglucose isomerase enzyme will be added to the assay mix to convert fructose-6-phosphate into glucose-6-phosphate and a second reading will be taken, the increase in absorbance will be used to quantitate fructose present. Assay mix components: Hexokinase Enzyme (HK), ATP, Glucose-6-Phosphate Dehydrogenase Enzyme (G-6-P DeH), NADP+, and Phosphoglucose Isomerase Enzyme (PGI) Assay chemistry: Two step coupled assay 1. D-Glucose + ATP→(HK)→G-6-P + ADP 2. D-Fructose + ATP→(HK)→F-6-P + ADP 3. G-6-P + NADP+→(G6P DeH)→gluconate-6-phosphate + NADH 4. F-6-P→(PGI)→G-6-P

• Fully quantitative because reaction equilibrium is far to the right.

Calculation: A = є•c•l Є=NADH molar extinction coefficient = 6.22 L/mMol•cm Example for glucose: A=0.628 c = A/є•l = 0.628/(6.22 L/mMol•cm)(1cm)= 101 µM Example for fructose: ∆A=0.739 c = A/є•l = 0.793/(6.22 L/mMol•cm)(1cm)= 127 µM

• Fructose levels will exceed glucose levels because fructose is a natural byproduct of cyanobacteria. Therefore this will be taken into account by testing wild-type cyanobacteria media.

Quantification of Ethanol Secretion Secretion will be tested using direct E.coli media samples and an alcohol oxidase assay. Assay Description: Alcohol Oxidase converts primary alcohols like ethanol and diatomic oxygen into a formaldehyde and a peroxide, respectively. The peroxide is then converted into two molecules of water by a peroxidase using an ABTS substrate as an electron donor. The resulting oxidized ABTS will absorb at 405nm. There is a 1:1 ration of ethanol to oxidized ABTS molecules; therefore we can use the molar extinction coefficient of oxidized ABTS in order to quantitate the amount of ethanol originally present. Assay mix components: Alcohol Oxidase Enzyme (A.O.), Peroxidase Enzyme (POD), ABTS (Azino-bis-(3-Ethylbenzothiazo line-6-Sulfonic Acid) substrate. Assay chemistry: Two step coupled assay 1. Ethanol + O2 →A.O.→formaldehyde + H2O2 2. H2O2 + ABTS→POD→2H2O + Oxidized ABTS

• Fully quantitative

Calculations: A = є•c•l Є=ABTS millimolar extinction coefficient = 36.8 L/mMol•cm Example: A=0.654 c = A/є•l = 0.654/(36.8 L/mMol•cm)(1cm)= 17.77 µM

Quantification of Fatty Acid Secretion Fatty acid secretion was determined using the EnzyChrom Free Fatty Acid Assay Kit from Bioassay Systems according to the manufacture’s protocol. Assay Description: This kit uses as one step assay in which fatty acids are enzymatically converted to acyl CoA and then to peroxide. The resulting peroxide reacts with a dye to form a pink colored product with O.D. at 570 nm. There is no extinction coefficient for this colored product a standard curve my be created in order to obtain a linear equation that can be used to determine unknown concentrations.

• For further assay information view the Bioassay Systems’ Free Fatty Acid Assay Kit manual.

Standards Used: Palmitic Acid standards of the following concentrations: 1000µM, 600µM, 450µM, 300µM, 200µM, 100µM, and a blank standard (no palmitic acid). These standard were used to create a standard curve by plotting [Palmitic Acid] against ∆A @ 570nm (∆A=standard absorbance – blank absorbance, or background). The standard curve was then used to give a linear equation of Y=mx+b. This equation can then be used to determine unknown sample concentrations by plugging the absorbance of the unknown in for Y and solving for x.

Apparatus

The E. coli is gravity fed to the transfer pump inlet, then pumped through clear vinyl tubing to the dialysis tube and returned back to the E. coli chamber. A variable four-channel aquarium oxygen pump, inside the apparatus base, oxygenates the Cyanobacteria and E. coli. Each chamber is fed from the bottom through two lines and check valves. On the top of the base is the adjustment knob to control the oxygen flow. Bubblers were attached to the top of each chamber to allow proper venting without releasing the bacteria out. The dialysis tubing is connected to a glass tube that is in the center of the Cyanobacteria chamber and secured with a shrinktube o-ring seal. The lighting is two T5 14W fluorescent bulbs, each connected to a slider that allows them to be independently adjusted. The base is constructed of 6 panels of 1/8 inch thick 6061 aluminum tig(GTAW) welded together. Its dimensions are approximately sixteen inches long, fourteen inches wide, and three inches tall.

Results

Figure 1. Confirmation of invertase operon parts. PCR was performed to amplify petBD+RBS, KanR, and inv with 20 bp flanking overlap regions. Equimolar volumes of each PCR product were mixed and ran on 0.7% agarose gel at 110 V for 60 minutes (lanes 2-9). 0.5 ug Kb+ standard was loaded into lanes 1 and 10.

Figure 2. Confirmation of agp in TopoIIBluntPCR vector. Agp was ligated into TopoIIBluntPCR vector (Invitrogen). 0.25 ug of each ligation was digested with SpI and PstI and ran on 0.7% agarose gel at 110 V for 60 minutes (lanes 2-8). 0.5 ug Kb+ standard was loaded into lanes 1 and 9. Lanes 3,5, and 8 confirm successful ligation.

Figure 3. Agarose gel electrophoresis of cleaved and uncut thiamin monophosphate pyrophosphorylase (ThiE) DNA. 0.5 ug of DNA samples were first isolated before a small portion was cleaved using EcoRI and Pst I restriction enzymes. Four cut samples and uncut DNA was separated by electrophoresis in a 1% agarose gel and visualized under a UV light. The results show a successful ligation of ThiE.

Figure 4. Agarose gel electrophoresis of PCR amplified chloramphenicol acetyltransferase (CmR) DNA. 5 ng of DNA was first amplified using PCR. The DNA product was separated by electrophoresis in a 1% agarose gel and visualized under a UV light. The results show a successful amplification of CmR.

Figure 5. Agarose gel electrophoresis of PCR amplified photosynthetic cytochrome b6 and subunit 4 (petBD) DNA. 4 ng of DNA was first amplified using PCR. The DNA product was separated by electrophoresis in a 1.5% agarose gel and visualized under a UV light. The results show a successful amplification of petBD.

Figure 6. Agarose gel electrophoresis of PCR amplified Zymonomas mobilis glucose facilitative transporter (GLF) DNA. Three different samples of 10 ng of GLF DNA was first amplified using PCR. The DNA products was separated by electrophoresis in a 1% agarose gel and visualized under a UV light. The results show a successful amplification of GLF.