Team:NU Kazakhstan/Results

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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.


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

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)

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.

Figure 10. Chromium reduction assay samples and controls.

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 11. 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 12. CPEC for all parts.

    Figure 13. Second trial of CPEC for three parts.

    Figure 14. Successfully transformed parts after CPEC

Validation of Part

Note: We struggled to optimize the protocol for Chromate reduction assay for about 3 months by changing the mass of variables and conditions, such as filtration, a method of protein extraction, an addition of protease inhibitors, keeping strict low-temperature conditions, pH adjustments and so on. The final results were obtained in the very last days.
There were three controls in the experiment: strains without chromium (background correction), Sodium acetate with chromium and NADH with chromium.
Chromate reduction assay performed by use of two strains of algae C.reinhardtii, namely with cell wall (cw+) and without (cw-). The measurement of Cr(VI) concentration as a period of time was conducted according to 1,5-DPC method described in Chromate reduction assay protocol. Non-electroporated algae samples (without Chromate Reductase) and electroporated algae (with Chromium reductase) were used. NADH substrate for chromium reductase was tested as well.
As it can be seen from the graphs 1 and 2, the natural ability of ability of C.reinhardtii to reduce Cr(VI) to Cr(III) was observed. However, there is a noticeable increase in reduced ability of electroporated samples of cw+ strains (almost two-fold decrease in hexavalent chromium) due to the presence of the recombinant protein - chromium reductase.

Graph 1. Reduction of Cr(VI) as a period of time by CW+ C.reinhardtii strain in the presence of NADH.

Graph 2. Reduction of Cr(VI) as a period of time by CW+ C.reinhardtii strain in the absence of NADH.

Considering the reduction of hexavalent chromium in the electroporated cw- stains from graphs 3 and 4, it is possible to say that while in graph 3 there was approximately 20% decrease in Cr (VI) concentration, in the graph 4 - about 12%. It could be proposed that the addition of NADH influenced the reduction ability of the cw- strain. Reduction ability of electroporated CW- strains upon the addition of NADH was not as dramatic as in the electroporated cw+ strains. The natural ability of cw- strains to reduce Cr (VI) upon the addition of NADH constituted much higher reduction as compared to the NADH- control. It could be suggested that this happened due the influence of the NADH in the mutant strain of C.reinhardtii.

Graph 3. Reduction of Cr(VI) as a period of time by CW- C.reinhardtii strain in the presence of NADH.

Graph 4. Reduction of Cr(VI) as a period of time by CW- C.reinhardtii strain in the absence of NADH.

In the graph 3, it can be observed that experiment with CW- (NADH+) was done improperly due to human error, because reduction ability of electroporated and non-electroporated cells are almost the same.
Overall, according to the results of the experiment, it was observed that the presence of Chromate Reductase substantially improved the efficiency of hexavalent chromium reduction for C. reinhardtii.

We have experimentally proven that the part central to our project works as expected.