Team:Nevada/Project/Ecoli

Contents 1 Project Introduction 2 E. coli Project 2.1 Production of Medium Chain Fatty Acids as a Biofuel 2.2 Production of Ethanol as a Biofuel 2.3 Construction of a Glucose Insensitive Inducible Promoter in E. coli

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.

E. coli Project

The goal of the E. coli team was to transform E. coli with genes that produce medium chain fatty acids and ethanol, both of which can be used as biofuels. In addition, the group wanted to develop an E. coli glucose insensitive inducible promoter that would not be down-regulated in the presence of high glucose concentrations. In our system glucose levels produced by Cyanobacteria may be unpredictable; therefore having an E. coli inducible promoter that is glucose-insensitive will provide more control over expression of the biofuel genes.

Pathway for Producing Biofuels and Ethanol in E. coli

Production of Medium Chain Fatty Acids as a Biofuel

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Bay Laurel Thioesterase Cloning Strategy
Bay Laurel thioesterase (BTE) is a gene that is naturally found in the bay leaves (Umbellularia californica)and used as a spice in cooking. The BTE produces medium chain 12-carbon and 14-carbon fatty acids (Voelker et al.,1996). 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 common in E. coli. The newly synthesized intermediate part (BBa_K558003) also contained an E. coli ribosome binding sequence, a His tag sequence on the 3’ end of the coding sequence and an iGEM registry double terminator sequence (BBa_B0014).

The constitutive promoter sigma 70 from the Registry of Standard Biological Parts (BBa_J23101) 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.

Free Fatty Acid Activity
Fatty Acid production was tested using EnzyChrom Free Fatty Acid Assay Kit (BioAssay Systems). The graphs below show the concentration of free fatty acid produced by Bay Laurel Thioesterase transformed in NEB 10 β and NEB Iq cell strains.

The NEB Iq E. coli cell line transformed with the sigma 70-BTE generator produced 125uM free fatty acid compared to the negative control sigma 70-red fluorescent protein (50uM) and the media only control (40uM). Bay Laurel Thioesterase transformed into NEB 10 β cells showed a positive increase of 115uM of fatty acids compared to negative controls of 40uM and 70uM. Based on this data we can produce up to 2.5 fold more free fatty acids with cell lines producing the Bay Laurel Thioesterase then without.

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Preliminary tests for fatty acid length showed C-12 in the Bay Laurel Thioesterase transformed cells, but more chromatography test need to be done in order to confirm the production of C-12 and C-14 medium chained fatty acids.

Production of Ethanol as a Biofuel

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Pyruvate Decarboxylase and Alcohol Dehydrogenase Cloning Strategy In order to produce ethanol efficiently in E. coli, is was necessary to transform E. coli with both the pyruvate decarboxylase gene to produce an acetaldehyde intermediate, and an alcohol dehydrogenase gene that will take acetaldehyde and convert it to ethanol. The Nevada team designed and synthesized an operon without a promoter (intermediate part k558000) containing pyruvate decarboxylase and alcohol dehydrogenase coding regions along with a standard E. coli ribosome binding site and double terminator (BBa_B0014). 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 (Ingram 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 concluded that the NEB T7 Express Iq cells produced higher levels of the acetaldehyde intermediate.

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 increasing amounts of glucose does not lead to higher ethanol production. It is possible that these lines are only producing low levels of alcohol dehydrogenase and we are seeing a buildup of acetaldehyde in these same lines. It may be necessary to re-evaluate the original operon and re-engineer it to improve the adh gene thereby increasing production of the alcohol dehydrogenase enzyme.

Construction of a Glucose Insensitive Inducible Promoter in E. coli

Although the constitutively expressed Escherichia coli sigma 70 promoter (J23101) has proven to be adequate for expression of our fatty acid and alcohol genes, the hope was to construct a new promoter for the iGEM registry that was inducible, but not sensitive to glucose or other sugars. The inducible promoter, trc, was a promising option. This promoter originated as a fusion between the trp (tryptophan inducible/glucose repressible) promoter and the lac (lactose inducible/glucose repressible) promoter (De Boer et. al. 1983). The trc fusion promoter was engineered in such a way that gene expression can be up-regulated in the presence of IPTG, but expression is unaffected by fluctuations in glucose concentration.

In spite of the encouraging research, trc has proven to be a complicated promoter to work with in the lab. With only 62 base pairs it is hard obtain definitive visual results and manipulate such small fragments between plasmids, cells, etc. There has been little success with this small fragment. Many techniques have aimed at transforming trc into pSB1C3 iGEM plasmid for submission to the registry for use by future teams and placing the promoter in front of both genes in various plasmids including pUC vectors, TOPO vectors, as well as iGEM vectors. Different techniques, enzymes, and protocols were used without success. One of the biggest challenges when using trc is that agarose gel electrophoresis is of little use for quick verification. The 100bp (promoter plus iGEM prefix and suffix) shift is nearly impossible to visualize on the gel and sequencing is often needed to confirm results which takes a few days to obtain results. Despite the frustration that we have had using the trc promoter, we think it would be a valuable part for the registry.