Difference between revisions of "Team:NU Kazakhstan/Results"

Vector construction

To absorb hexavalent chromium, to hold it inside the cell and to reduce it to less toxic trivalent form, four gene expression cassettes were placed into pChlamy_4 acceptor vector.
Four genes, AphVIII, ChrR, Chromodulin and Membrane-bound SuperNOVA were expressed under fused pHSP70+pRBCS2+Intron (RBCS2) promoter.

OVERVIEW

1. Hexavalent Chromium Resistant Strain Of C.reinhardtii

   • C.reinhardtii were transformed with PJD67 to induce mutagenesis in the algae and eventually obtain chromium (VI) resistant strain.

2. Combination Of All Parts Into Two Expression Vectors

   • Two constructs, namely Chromodulin and ChrR, were placed into pHyg3 expression vector.
   • Chromodulin+ChrR were placed to pChlamy_4 vector with an endogenous promoter for C. reinhardtii.

3. Transformation Of C.reinhardii

   • pHyg3 with Chromodulin+ChrR was electroporated into C.reinhardtii.
   • pChlamy_4 with Chromodulin+ChrR was electroporated into C.reinhardtii.

4. Verification Of Transformation Of C.reinhardtii And Expression

   • Confirmation of the presence of the genes in the colonies was done.
   • Expression of …… was checked via Western Blot.

5. Hexavalent Chromium Uptake And Reduction Ability examination

   • Ability of transformed C.reinhardtii to absorb hexavalent chromium was checked.
   • Ability of transformed C.reinhardtii to reduce hexavalent chromium was checked.

I. Hexavalent Chromium Resistant Strain Of C.reinhardtii

Electroporation of the algae with pJD67 vector was done. This gives C.reinhardtii an ability to live in non-arginine media. pJD67 transformation allowed us to induce mutagenesis and get Chlamydomonas reinhardtii able to live under 0.05mM and 0.1mM Cr(VI) were obtained.

Figure 1. C.reinhardii transformed with pJD67 grown under 0.05mM and 0.1mM hexavalent chromium.

II. Combination Of All Parts Into Two Expression Vectors

Two of four cassettes were placed into newly obtained strain of C.reinhardii. SuperNova was not transformed into algae, since of the toxicity of natural light to it and AphIII gene was synthesized incorrectly, so it was inapplicable.
Chromodulin and Chromate Reductase genes were successfully placed placed into our C.reinhardii.

III. Transformation Of C.reinhardii

Figure 2. PCR of transcriptional units of ChrR, SuperNova and Chromodulin amplified from pHYG(ChrR+Chromodulin).

Figure 3. pHyg3 (ChrR+Chromodulin) electroporated C.reinhardtii.

Figure 4. pChlamy_4 transformed C.reinhardtii.

IV. Verification Of Transformation Of C.reinhardtii And Expression

The integration of pHyg plasmid and pChlamy was verified using PCR for both colonies and liquid cultures.
Positive control - PCR run on pHyg plasmid with primers for chromate reductase (ChrR)
Sample - either liquid culture or a colony

V. Hexavalent Chromium Uptake And Reduction Ability examination

The integration of pHyg plasmid and pChlamy was verified using PCR for both colonies and liquid cultures.
Positive control - PCR run on pHyg plasmid with primers for chromate reductase (ChrR)
Sample - either liquid culture or a colony