Coding

Part:BBa_K2516000

Designed by: Valdemir Kim   Group: iGEM17_NU_Kazakhstan   (2017-10-25)
Revision as of 03:27, 2 November 2017 by Valdemirkim (Talk | contribs)


Chromate reductase (codon-optimized for C. reinhardtii)

Chromate reductase (ChrR) is an oxidoreductase enzyme that is able to transfer 3 electrons towards Cr6+ anions, which results in reduction directly into Cr3+ ions. This reduction avoids production of side products such as Cr5+ and Cr4+, which are quite toxic to the cell. Both NADH and NADPH are equally utilized as electron donors. Other substrates that can be reduced: quinones (primary biological role), potassium ferricyanide, 2,6-dichloroindophenol, V5+, Mo6+, methylene blue, cytochrome c and U6+.


Reaction of electron transfer is the following:

2 NAD(P)H + Cr6+ + O2 = 2 NAD(P)+ + Cr3+ + superoxide


Flavin mononucleotide (FMN) and Ca2+ serve as cofactor molecules.

This enzyme lacks any post-translational modifications.


Optimal Conditions:

Optimum pH (for Cr6+) = 5

Optimum temperature (for Cr6+) = 35 degrees Celsius

Validation

Chromium 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 1,5-DPC method described in Chromium reduction assay protocol. Non-electraporated algae samples (without Chromate Reductase) and electraporated 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 reduction ability of electraporated samples of cw+ strains (almost two fold decrease in hexavalent chromium) due to the presence of the recombinant protein - chromium reductase.


NADH%2B%28CW%2B%29.jpeg
Graph 1. Reduction of Cr(VI) as a period of time by CW+ C.reinhardtii strain in the presence of NADH.


NADH-%28CW%2B%29.jpeg
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 electraporated 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 electraporated CW- strains upon the addition of NADH was not as dramatic as in the electraporated 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.


NADH%2B%28CW-%29.jpeg
Graph 3. Graph 3. Reduction of Cr(VI) as a period of time by CW- C.reinhardtii strain in the presence of NADH.


NADH-%28CW-%29.jpeg
Graph 4. Reduction of Cr(VI) as a period of time by CW- C.reinhardtii strain in the absence of NADH.

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.


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 319
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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