Team:Caltech/Project

Overall Project

Lignin, cellulose, and hemicellulose comprise lignocellulose, a critical component of plant cell walls. Its stability contributes to the rigidity of the plant cell walls and the plants themselves. Hemicellulose is relatively easy to break down compared to cellulose and lignin, because its building blocks are mostly shorter sugar chains. However, lignin and cellulose degradation remain high-energy endeavors which block efficient biofuel production. Select fungi, as well as gut microbes of certain termite species, degrade these organic polymers. Our hope is to isolate these genes and introduce them to E. coli, so that given plant biomass, a much higher percentage of the hydrocarbons would be converted to fuel.

Alginate is a product of certain infectious bacteria, like Pseudomonas aeruginosa. Alginate forms a biofilm around the invader colonies in the lungs, which limits antibiotic effectiveness. However, researchers have found E. coli and S. aureus to react more adversely to antibiotics when exposed to sugar; we will determine if the same can be said for P. aeruginosa by breaking down the alginate biofilms into simpler sugars. We hypothesize that this will both allow antibiotics ease of access to the target colonies and make the bacteria more susceptible to the drugs. Our approach to alginate breakdown parallels that of lignocellulose degradation.

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 will introduce proteorhodopsin and a cofactor, retinal, to E. coli and aim 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, we will 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

The idea behind this project to transform Z. mobilis so that it is able to ferment on substrates outside of what it naturally consumes. There are two parts to this project.

In the first part of the project, we need 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 will electroporate the Z. mobilis cells and then grow the transformants on the substrate as the sole carbon source. Electroporation, growth, and cryopreservation protocols are shared on GoogleDocs.

In the second part of this project, 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

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.