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. Combination Of All Parts Into Two Expression Vectors

   • Two constructs, namely Chromodulin and ChrR, were placed into pHyg3 expression vector.
   • ChrR and SuperNova were amplified using plasmids which add restriction sites for further cloning into pChlamy_4 vector with an endogenous promoter for C. reinhardtii
     

     Figure 1. PCR of transcriptional units of ChrR, SuperNova and Chromodulin amplified from pHYG(ChrR+Chromodulin).

   • Amplified fragments were digested and ligated with pChlamy vector, followed by transformation to DH5-alpha
     

     Figure 2. Successful transformation of ligation product of pChlamy+SN, pChlamy+ChrR

2. Transformation Of C.reinhardtii cell wall+ strain

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

     Figure 3. C.reinhardtii transformed with pHyg

   • C.reinhardtii cell wall+ strain electroporated with pChlamy_4 +ChrR
     

     Figure 4. C.reinhardtii transformed with pChlamy + ChrR

   • C.reinhardtii was electroporated with pChlamy_4 +SuperNova

     Figure 5. Transformed C. reinhardtii with pClamy + SuperNova was kept under red-blue light, which doesn’t contain 585 nm (excitation wavelength)

3. Verification Of Transformation Of C.reinhardtii And Expression

   • Confirmation of the integration of plasmid into genome was done using colony PCR and PCR on liquid cultures.
     

     Figure 6. PCR of ChrR from liquid cultures and colonies

   • Expression of Chromate reductase protein was checked via Western Blot.

   • 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.

   • Hexavalent Chromium Resistant Strain Of C.reinhardtii

     C.reinhardtii was transformed with pHyg and pChlamy to create resistant strains. We successfully created strains resistant to 0.05 mM and 0.1 mM chromium concentration. Also we produced fully photosynthetic cell wall deficient strain, that was cultured in TAP minimal without acetate (main carbon source in TAP medium) resistant to 0.05 mM chromium. To compare, maximum concentration of chromium found in rivers and lakes of Kazakhstan is 0.017 mM. Our strain has ability to survive even higher concentrations, therefore it can be potentially applied to real conditions.
     Electroporation of the algae with pHyg vector with Chromate reductase was done. This gives C.reinhardtii an improved ability to survive in chromium-containing medium compared to control. pHyg transformation allowed us to induce mutagenesis and get Chlamydomonas reinhardtii able to live under 0.05mM and 0.1mM Cr(VI) were obtained.

     
     

     Figure 7. Strain resistant to 0.05 mM chromium

     
     

     Figure 8. Mutant strain resistant to 0.1mM hexavalent chromium.

     
     

     Figure 9. Fully photosynthetic strain, electroporated with pHyg.

   • Submission of parts.

     • We amplified ChrR and Chromodulin parts from pHyg plasmid and SN, promoter and terminator were amplified from SN transcriptional unit. Primers were designed to add restriction sites for further Circular Polymerase Extension Cloning (CPEC)
       

       Figure 10. PCR of parts for submission.

     • We successfully cloned all of our parts into pSB1C3 vector using CPEC.
       • We had two trials. In the first run we got the following gel image (Figure 11). All of the parts were cloned successfully, however transformation of DH5-alpha was successful only for 2 parts: terminator and SuperNova
       • We repeated experiment for other three parts: ChrR, Chromodulin, promoter. (Figure 12) This time we also got positive results. Transformation of these parts produced colonies. (Figure 13)
       
       

       Figure 11. CPEC for all parts.

       
       

       Figure 12. Second trial of CPEC for three parts.

       
       

       Figure 13. Successfully transformed parts after CPEC

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