Team:Nevada/Project

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We are focused on creating a self sustaining biofuel production system. To accomplish this we must co-cultivate two different types of bacteria that will eventually lead to the production of ethanol and fatty acids used for biofuel. The idea is based upon using a photosynthetic bacteria to feed E. coli which will then produce the ethanol and fatty acids. This would drastically reduce the cost of production of biofuels, which is the main problem we have with them as a viable energy source at this time. This also has implications for the drug industry as many medicines, such as insulin, are manufactured from E.coli bacteria.
To get this system to work we must genetically engineer the different bacteria to allow us to change their natural production processes. With the photosynthetic cyanobacteria we will be incorporating 2 new genes as well as knocking out 2 that already exist in the bacteria. This will allow us to produce the maximum amount of glucose and secrete it outside the cell. The E.coli will then feed off this glucose and use it to grow and produce ethanol.
In engineering E.coli we will be incorporating 3 genes. PDC/ADH and BTE. These are genes that regulate and control metabolic pathways and will lead to the production of ethanol and fatty acids respectively. Our two bacteria will then be grown together in a new system that we have designed to allow for the exchange of glucose through a semi-permeable membrane. E.coli will feed off this glucose while producing ethanol and biodiesel which we can extract from our production chamber.

Introduction

Abstract:

Introduction:

Approach

E. coli

Fatty Acid Production

Ethanol Production

-Engineered Pyruvate Decarboxylase and Alcohol Dehydrogenase coding regions (based on Zymomonas mobilis, a microorganism that naturally produces ethanol).

- Removed PDC/ADH from pUC57 vector and put into pSB1C3 -Sequenced and confirmed PDC/ADH in pSB1C3 -Submit PDC/ADH/pSB1C3 to iGEM

-Put a constitutive promoter (σ70) in front of PDC/ADH genes to test expression of ethanol. -Opened up σ70 in Amp vector, then ligated with PDC/ADH. -Sequenced and confirmed σ70/PDC/ADH in Amp vector.

With σ70/PDC/ADH in Amp vector (in NEB 10β cells): -Performed Ethanol Detection test- no samples produced detectable ethanol above background… -Performed ADH Enzymatic test- no ADH detected in assay.

-created “Aldehyde Detection Plates” to test PDC presence & functionality -re-transformed σ70/PDC/ADH in Amp vector into NEB Iq cells -Plates & colonies turn Bright pink- so Aldehydes ARE present (possibly being secreted)

-When ethanol can be detected from σ70/PDC/ADH in Amp vector, I will get -σ70/PDC/ADH into pSB1C3 and submit as an iGEM part. -We are still working on getting pTRC in front of PDC/ADH in pSB1C3

E. coli Results

Cyanobacteria

The goal of this project is to engineer the cynaobacterium 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

1) 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. Approach 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 Design:

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.

petBD promoter+RBS: The strong light inducible petBD promoter will be used to drive the expression of invertase gene in Synechocystis. The petBD+RBS (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

1. 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.

2. 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.

3.Assembly of Parts:

The above parts will be assembled into the following construct:

Transformation: Synechocystis will be naturally transformed via homologous recombination.

ThiE/glF Operon

The ThiE gene in Cyanobacteria is responsible for thiamine production in the cell. This is essential for cell survival. By knocking out this gene, we ensure that the Cyanobacteria cannot survive outside of media that provides this substance,and thus it serves as our environmental control.
The ThiE knockout will be accomplished by inserting the genes of interest into the ThiE sequence. The inserted genes will be expressed so that the genetically altered Synechocystis can produce glucose in a controlled environment. The gene operon is as follows: Chloramphenicol resistance for selection of desired species in liquid cultures and on plates; petBd, a promoter to direct products outside of the cell; and glF, a glucose facilitator/transporter. The finished construct looks like this:
(A couple sentences explaining where each of these genes came from is needed, I need to double check the official names of the cells before writing it here. If you have them somewhere, feel free to add it. Otherwise I will get the info on Sunday)
The genes of interest were prepared by designing custom primers which were then amplified through PCR. Proper amplification of DNA was ensured through electrophoresis in agarose gels. Bands in the agarose gel that displayed the correct behavior were extracted and purified using QIAGEN mini-prep kits. PCR products were also submitted for genomic sequencing and BLAST to confirm that they contained the correct DNA. After successful isolation of each gene, they were combined using Gibson Assembly. This method was described in the above heading, titled AGP/inv Operon. Once the GLF operon was complete, it was then inserted into the ThiE sequence that naturally occurs in Synechocystis. Selection for the transformed colonies was preformed through screening with chloramphenicol resistance.

Media Development

Assay Development

Methods 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 6061 aluminum tig(GTAW) welded together. Its dimensions are approximately sixteen inches long, fourteen inches wide, and three inches tall.