Part:BBa_K2242233

Cysteine Desulfhydrase

Cysteine Desulfhydrase is an aminotransferase that converts cysteine into pyruvate, ammonia, and hy- drogen sulfide. Because cysteine desulfhydrase activity is not restricted to anaer- obic conditions, expression of cysteine desulfhydrase in a suitable host could result in aerobic precipitation of cadmium as cadmium sulfide. (Wang, C., Lum, A., Ozuna, S., Clark, D., & Keasling, J. (2001). Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene. Applied microbiology and biotechnology, 56(3-4), 425-430.) In our project, we need to use CdS nanoparticles to generate electrons utilizing light energy. So we need to find a way to produce CdS nanoparticles on the membrane of our engineered E.coli. The easiest way is to precipitate those nanoparticles directly, using the sulfide ions the bacteria produce and the cadmium ion we add into the reaction system. We introduce this enzyme to produce sulfide ion in the cytoplasm of our E.coli efficiently.

Introduction

This year, we improved the characterization of a part encoding the cysteine desulfhydrase(BBa_K731400) from team iGEM12_TRENTO. We found out a new function of the part, to generate sulfur ions from cysteine to precipitate the cadmium ions. In another word, this protein can increase the bacteria's resistance to a certain concentration of cadmium ions and solve the pollution of cadmium ions. With this part, we can successfully precipitate CdS nanoparticles on the surface of our bacteria.

The reason we chose this part for contribution is that we found out that many characterizations should be added if we want to have a thorough understanding of this part. For example, although it can precipitate cadmium ions to increase the resistance, but what is the maximum concentration of cadmium ions it can take? We did a growth curve test to find it out. Besides, we also used Transmission Electron Microscopy(TEM) to confirm that the precipitations are form on the surface on the bacteria because it needs to attach to the bacteria to function in our project.

1.CysDes-pLuxR-pSB1C3 construction and transformation

We obtained the sequence of CysDes gene from Genebank and synthesized this gene from IDT. We inserted this gene to plasmid pSB1C3 with promoter pLuxR on it which was provided by iGEM headquarters. The sequence of gene CysDes was validated with DNA sequencing by Sangon. We transformed this plasmid plays (one contains gene CysDes and promoter pLuxR) into strain BL21. Then we picked some colonies for cultivation and confirmed the transformation result by PCR (shown in Figure 1). From the result of electrophoresis, we confirmed the transformation of pLCys was success.

Figure 1. Electrophoresis result of bacterial PCR (positive result: Cys+pLuxR-1 Cys+pLuxR-2 negative result:Cys+pLuxR-3 Cys+pLuxR-4)