Team:Caltech/Project

Overall Project

Our overall project is do degrade things like lignin and alginate and generate biofuel. Our notebook is organized by project. Below we summarize each project.

Degradation Project

Degradation summary: we degrade things.
Degradation Notebook

Proteorhodopsin 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 [].

Biofuel 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”.

Coliroid Project

Summary of coliroid project.