Difference between revisions of "Part:BBa K2242233"

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        <h3>Introduction</h3>
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                                                        <p class="indent_word">This year, we improved the characterization of a part encoding the cysteine desulfhydrase(<a href="https://parts.igem.org/Part:BBa_K731400">BBa_K731400</a>) 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.
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                                                        <p class="indent_word">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.
 
                                                         <h3>1.CysDes-pLuxR-pSB1C3 construction and transformation</h3>
 
                                                         <h3>1.CysDes-pLuxR-pSB1C3 construction and transformation</h3>
 
                                                         <p class="indent_word">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. </p>
 
                                                         <p class="indent_word">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. </p>

Revision as of 10:36, 30 October 2017


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)



2.Expression of CysDes

We inoculate 2 mL overnight culture to 200 mL LB media (1mM cysteine, 30mM glucose and 10mM HEPES are included) and cultivate for 2h at 37 ˚C. When OD600 reaches 0.4-0.6, add AHL to final concentration of 250nM. After 3h cultivation, collect the bacteria by centrifugation. Then extract the raw enzyme of CysDes by ultrasonication. We run SDS-PAGE of samples of raw enzyme, cell content obtained by 100 ˚C heating and wild type (shown in Figure 2). The protein CysDes is about 46kDa, we can find obvious bands at the about position of 45kDa which are unique to lanes of cell contents after induction and raw enzyme compared with the wild type. Although there are proteins of similar molecular weight in wild type, darker bands in experiment group meaning a high amount of proteins could prove the existence of high amount of CysDes. From the result of SDS-PAGE, we could confirm the expression of CysDes.

Figure 2. SDS-PAGE of cell lysate

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 297
  • 1000
    COMPATIBLE WITH RFC[1000]