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Team:Caltech/Project
From 2012.igem.org
Overall Project
Many marine proteobacteria possess a light-activated transmembrane proton pump called proteorhodopsin. Normal E. coli strains use NADH to create a proton gradient; however, in fuel-making cells, NADH is an important component of the synthesis reaction. If we could manipulate the E. coli to rely mostly or solely on proteorhodopsin as a proton pump, it would be much easier to synthesize biodiesel. We introduced proteorhodopsin and a cofactor, retinal, to E. coli and aimed to make the ATP synthesis mechanism completely dependent on the proteorhodopsin-established proton gradient, freeing NADH to act solely in another synthesis pathway. To ensure dependence on the new mechanism, in the future we hope to knock out the pathway that utilizes NADH to create the proton gradient.
Generating biodiesel is an energetically demanding process. Therefore, in addition to proteorhodopsin, we plan to knock out the fermentation pathways of E. coli which yield byproducts such as lactase and succinate. Fermentation uses NADH, the reducing agent also essential for the biodiesel synthetic pathway. E. coli can generate ATP anaerobically or aerobically; its fermentation pathway reduces pyruvate to a variety of products, including lactase and succinate. We want to eliminate this reduction pathway because alkyl esters also require reduction during formation. The more NADH available to assist in reduction during fatty acid synthesis, the higher our alkyl ester yield will be.
Degradation Project
We pursued multiple routes to develop possible degradation pathways for our compounds. One route involves conducting “gene fishing” experiments to isolate organisms that are capable of degrading our compounds and from them isolating the genes responsible. It’s possible to isolate an organism that can subsist on our compounds by taking samples from the environment and growing them in a medium in which the sole source of carbon is the compound we wish to degrade. Specifically, this method involves growing bacterial cultures in liquid minimal media and sequentially inoculating new cultures in order to further dilute the sample, and then plating onto solid minimal media (in order to establish that the organism is in fact degrading the compound and not simply living off of trace amounts of, for example, carbon dioxide). If an organism can grow in such a medium, then they must have a mechanism to degrade the compound, and we can then isolate the genes responsible for degradation.
The other facet to the degradation project is transforming Z. mobilis so that it is able to ferment on substrates outside of what it naturally consumes. There are two parts to this subproject.
In the first part of the subproject, we needed a plasmid containing the gene that codes for a degradation enzyme. The vector must be Z. mobilis compatible. We will be getting this vector from the Oak Ridge National Laboratory. The DNA insert must not have more than 4 BioBrick restriction sites. Then, we electroporated the Z. mobilis cells and then grew the transformants on the substrate as the sole carbon source.
In the second part of this subproject, we will use a certain type of plate from Biolog. The Biolog plates contain many wells; each well containing a different carbon source. We will suspend E. coli and Z. mobilis cells in the wells of the Biolog plates. We expect to see that E. coli can grow in wells that Z. mobilis can not and vice versa. We will also expect to see some overlapping wells. We will then digest the E. coli bacteria, and then use those digest fragments as DNA inserts to be transformed into Z. mobilis. Because the DNA fragments will be very large, which will create very large plasmids, we can not simple electroporate the plasmids directly into Z. mobilis. So instead, we will need to use a kit to use a bacterial phage and insert the plasmid into E. coli. Then we can transfer the plasmid from the E. coli to Z. mobilis by conjugation. We will then expect the transformed Z. mobilis to be able to grow in the wells that E. coli and the original Z. mobilis Biolog plates.
Degradation NotebookProteorhodopsin Project
The production pathways we plan to introduce in ''E. coli'' require NADH for the reactions; however, ''E. coli'' require NADH to donate protons and generate the proton-motive force that drives its ATP synthase to produce ATP. ''E. coli'' uses NADH dehydrogenase to convert NADH to NAD+ and expel the proton outside of the cell membrane. We plan to make the production pathways by reducing the bacteria's inherent need for NADH in two ways: 1. Lambda Red removal of Nuo, an NADH dehydrogenase found in ''E. coli''; 2. introduction of proteorhodopsin, a light-powered proton pump, into ''E. coli'' to replace the electron transport chain.
When testing the effects of proteorhodopsin in ''E. coli'' with the proteorhodopsin gene added and nothing removed, we realize that ''E. coli'' will not make use of proteorhodopsin under normal conditions, since the electron transport chain is more optimal for ATP production. Thus, we must grow ''E. coli'' under stressful conditions to induce it to utilize the pump.
Proteorhodopsin NotebookBiofuel Project
Biodiesel is made up of a variety of fatty acid alkyl and methyl esters, as well as long-chain mono alkyl esters. In principal, biodiesel is a great fuel source because after discarded hydrocarbons are transesterified (when an alcohol and ester swap R groups), the subsequent alkyl ester-based fuel burns more efficiently than “normal” diesel and reduces the wear on engines. Unfortunately, biodiesel is not a completely viable or reliable energy source because of low production yields. A team of researchers at Berkeley engineered a strain of E. coli capable of producing alkyl esters at 9.4% of theoretical yield, which is on the higher end of current yields of biologically derived alkyl esters. To make biodiesel cost competitive, we need to increase yield per substrate.
Generating large volumes of alkyl esters per substrate is an energetically demanding process; for this reason, one way we will increase yield is by incorporating a proteorhodopsin – dependent energy producing mechanism into the cells. However, we need as much NADH as we can for the synthetic pathway, and for this reason we will pursue methods to increase NADH concentration further. The biodiesel synthetic pathway consumes a large amount of NADH, a reducing agent. E. coli can generate ATP anaerobically or aerobically; its fermentation pathway reduces pyruvate to a variety of products, including lactase and succinate.. We want to eliminate this reduction pathway because alkyl esters also require reduction during formation. The more NADH available to assist in reduction during fatty acid synthesis, the higher our alkyl ester yield will be. There are a variety of ways to increase the NADH/NAD+ ratio in our E. coli cells. Our first step will be to knock out E. coli’s NADH dehydrogenase enzymes Nuo and Ndh, which typically oxidize NADH. E. coli’s genome has been entirely sequenced, so we can use lambda red recombination engineering to target the two enzymes’ genes. The general procedure is as follows. We will grow up our E. coli strain (which has minimal alkyl ester yield). We then will take the Nuo/Ndh homologous knockout genes and introduce the plasmids into the cells. During gene replication, some cells will transcribe the new (null) copy of the gene instead of their own. We will grow up colonies of our E. coli and determine which colonies have taken the null genes by PCR verification. This procedure should take about three weeks.
Once we isolate Nuo/Ndh deficient E. coli, our strain will have excess NADH. Because the NADH/NAD+ concentrations in E. coli should be balanced, the cells will need to compensate by reducing NADH some other way. We intend that the fatty acid synthesis and transesterification processes will consume more of these available electrons, thus improving yield of the target product and decreasing byproduct volume simultaneously. When we merge the proteorhodopsin project with the biofuel project, even more NADH will become available for synthesis. We will measure the volume of alkyl esters we produce using the GCMS (gas chromatography mass spectrometry) procedure, as specified in the paper “Isotope Abundance Analysis Method and Software for Improved Sample Identification with the Supersonic GC-MS”.
Biofuel NotebookBacterial Animation
For the bacterial animation project, we are working on creating a construct of a fluorescent protein (mCherry), with a degradation tag attached into a light sensing system in Escherichia Coli that will sense light and “photograph” a light pattern as a high-definition chemical image and then animate by the degradation tag. This project was a collaboration with students from Cal Arts, who will focus on the animation of these coliroid plates. Not only is this project fun, it has potential applications in specific temporal and spatial control of biofilms. In addition to publicizing synthetic biology, our project will satisfy one of iGEM’s requirements of human practices and outreach.
This concept of designing a bacterial system in coliroid that is switched between different states by light has been previously achieved with expression of a lacZ reporter. The system allows a lawn of bacteria to act as a biofilm, so that the projection of a pattern of light on the bacteria produces a high-definition chemical image. The primary focus is to achieve this same printing, but by using a fluorescent protein (mCherry) instead of the lacZ reporter. Also, we aim to animate the images produced by the bacterial film, by including degradation tags that will lower the intensity of fluorescent while light is being shone, and eliminate it when there is no light source. In this way, we hope to create an animation from these coliroid plates as the degradation tags degrade the image produced by the bacteria over time.
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