Crispr

Introduction

The mean function of carbon fixation E.pangu is transforming inorganic substances into organic substances. Unfortunately, the process will definitely be affected by their natural ability of utilizing glucose to serve energy to E.pangu themselves. Thus, our project includes a part in which we will knockout the genes of glycolysis to verify the functions of carbon fixation E.pangu. The gene circuit for this part is shown in figure 1:

Fig. 1. Gene circuit of carbon fixation testing

Here we introduced a gene editing technique called CRISPR Cas9 to knock out glycolysis of E.pangu. CRISPR is widely used to edit genome based on the complexity of sgRNA and Cas9 protein. Unlike other gene edition technique, CRISPR can be designed in one dimension instead of stereoscopy. Thus, when targeting different sites, we just need to design corresponding sequences. After knocking out several key enzymes, we will be able to regulate the metabolism of E.pangu, so that the ability of carbon fixation of our engineered microbe can be qualified.

At the initial stage of bacterial growth, the energy requirement is relatively high. To ensure the normal growth at this stage, we added a regulated promoter before Cas9 protein, through which the glucose catabolism of our bacteria can be regulated. That is, we will not knock out the glycolysis system until the cell growth has reached stationary phase.

Results

1.pRhl Insight

In this summer, we are going to design a system which is able to fix inorganic carbon and nitrogen simultaneously. This system consists of 2 types of E.coli: nitrogen fixation E.coli and carbon fixation E.coli. We hope this system will play the role of pioneer organism on Mars immigration. Based on this purpose, we call them E. pangu collectively, which is named after the creator of universe, Pangu in Chinese mythology, as they will reclaim Mars for future immigrated organisms.

Our project contains the following contents:

1.Nitrogen fixation E.pangu

Azotobacters are nitrogen fixation bacteria. They catalyze N2 reduction into ammonia by nitrogenase. The nitrogen fixation genome contains a large number of modules, or clusters. After deleting unnecessary modules[2], we will transform the minimal nitrogen fixation genome from azotobacter Paenibacillus sp. WLY78 into E.coli to construct nitrogen fixation E.pangu.

2.Carbon fixation E.pangu

Many anaerobes carry out carbon fixation by Wood-Ljungdahl pathway. One of the core enzymes in this pathway is acetyl-CoA synthetase/carbon monoxide dehydrogenase (ACS/CODH), a bifunctional enzyme which can reduce CO2 into acetyl-CoA, the central molecule in metabolism. We will transform the gene of ACS/CODH into E.coli to construct our carbon fixation E.pangu.

3.Testing

In this part, we will introduce gene editing technique called CRISPR-Cas9 to knock off the gene of pyruvate dehydrogenase(PDH) complex of carbon fixation E.pangu. Without PDH, the normal pathway of acetyl-CoA producing is cut off. Thus, if our carbon fixation E.pangu was successfully constructed, they can grow normally without PDH.

Reference

[1]Krasnopolsky, V A.; Feldman, P D. Detection of Molecular Hydrogen in the Atmosphere of Mars. Science, 2001, 294:1914-1917.

[2]Wang L, Liu Z, Zhao D, Liu X, et al. A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli. PLoS Genet, 2013, 9:e1003865.

[3]Lindahl P A. The Ni-containing carbon monoxide dehydrogenase family: Light at the end of the tunnel. Biochemistry, 2002, 41:2097-2105.