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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. We built our proteorhodopsin strain using PCA assembly based on the information from this paper . 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

View the Degradation Notebook

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.

Proteorhodopsin Project

View the Proteorhodopsin Notebook

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. There are a variety of ways to increase the NADH/NAD+ ratio in our E. coli cells. One of our objectives was 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 tried lambda red recombination engineering to target the two enzymes’ genes. We are still trying to knock out both genes in one strain of E. coli.

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. Another way we tried to increase yield is by incorporating a proteorhodopsin – dependent energy producing mechanism into the cells. The more NADH available to assist in reduction during fatty acid synthesis, the higher our alkyl ester yield will be.

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.

Bacterial Animation

View the Bacterial Animation Notebook

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. We obtained the fluorescent strain from the Murray Lab at Caltech. 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.