1-What is cytochrome C?
More than its biological functions, cytochrome c can catalyse several reactions such as hydroxylation and aromatic oxidation, and shows peroxidase activity by oxidation of various electron donors such as 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS).
2-How does the cytochrome C work in our project?
- 1-The correct distance between redox centres of the laccase and the cytochrome
- 2-The right orientation of both proteins
- 3-The consistent redox potential difference (ΔE) between donor and acceptor
3-What are our constructions
In each case, two types of construction have been done: a cytochrome c alone and a cytochrome c linked to a laccase. In both cases, we anticipated a problem to overcome. To be functional, a cytochrome c must integrate a heme into its structure. As we overexpressed the cytochrome C, we should overexpress simultaneously hemes. In this purpose, we chose to clone the heme A gene under the control of the T7 promotor (Bba_.......). This construction is carried by an independent plasmid.
Unfortunately, we did not manage to get the whole construction.
Purification of Cytochrom C
After sonication, we analyzed cell extracts by SDS-PAGE gel 15% (Gel1) to determine the localisation of the cytochrome C: in the supernatant or in the cell pellet.
Note that we produced the cytochrome C under two different conditions: in an aerobic (Aero) or in an anaerobic (Anaero) condition.
As we can see, the cytochrome c is located in the cell pellet, more precisely in the cell membranes. This result suggests us that the cytochrome C is associated to or inserted in the membrane. We solubilized the cytochrome C with detergent (triton) overnight 3 times. We did mistakes such as using the wrong detergent, the wrong SDS-PAGE gel or a not effective bad sonication that did not break cells…) Finally, we managed to analyze our western-blot. After solubilization, the cytochrome C is in low quantity in the cytoplasm and in the membrane extracts. We obtained more quantity in the solubilized membranes.
But we know that we can produce it. Maybe if we had more time, we could purify it and then test it on polymers.
Maybe it’s because we did not produce Heme A with our Cytochrom C.
Problem with the folding
Production of Laccase E.coli
Thanks to digestion, ligation and transformation, we managed to get our BioBrick named “01-35-02” with a size of about 1700 pb:
Figure A: Schematic representation of “01-35-02”
Production of laccase T.thermophilus from iGEM parts
Then we tried to add to this BioBrick a promoter and a His-Tag.
Unfortunately we managed to add only the promoter.
Figure D: Schematic representation of “01-36”
Production of laccase T.thermophilus from IDT
From this optimised laccase, we added a promoter and a His-Tag.
This new BioBrick is named “01-30-02” with an expected size of about 1500 pb
Figure F : Schematic representation of “01-30-02”
Purification of Laccase E.coli
01-3502 Laccase E.coliWe produced the E.coli Laccase. We compared non-induced and induced culture on a SDS-PAGE gel (15%) by Coomassie blue staining and in parallel by Western-Blot (anti-His).
We saw that the IPTG induction worked ! So we tried to purify it on Ni-NTA resin. Then we did another SDS-PAGE (15%) with flow through and eluted parts.
We successfully purified it ! But when we did our enzymatic tests on it, we had no activity. We think that maybe we should produce under anaerobic conditions. But we missed time to try it…
01-3002 Laccase TT optimizedThe construction was validated, we tried to purify it under anaerobic condition but we did not succeed…
Question 1: Can the laccase oxidize the cytochrome C?To answer this question, we used absorbance properties of the cytochrome C (FIG.1). Indeed, the reduced cytochrome C (curve in red) absorbs at 550 nm whereas the oxidized cytochrome C (curve in green) doesn’t absorb. By spectrophotometry, we analyzed the change in oxidation state. When the cytochrome C is alone, we don’t observe oxidation (FIG.2, blue curve). When we add the laccase, the cytochrome C oxidizes as we can see a decreased absorbance at 550 nm (FIG.2, red curve). We can see the effect of the laccase on the cytochrome C.
Conclusion 1: The laccase can oxidize the cytochrome C.
Question 2: Can the cytochrome C and the laccase oxidize chewing-gum polymer?
The aim of our project is to show that the chewing gum can be degraded by the laccase and the cytochrome C in presence of the light.
Using the X compound, the light and the laccase, the styrene that is a close chewing gum polymer can be oxidized (FIG.3 & FIG.4). The X compound is a rare metal so we don’t want to use it.
Could the cytochrome C substitute the X compound to oxidize the styrene?
To test our hypothesis, we used an oxygraph. This engine allows us to measure dioxygen concentration consumed during the reoxidation of the X compound or the cytochrome C by the laccase.
The laccase coupled with the X compound can oxidize the styrene (FIG.4). Replacing the X compound by the cytochrome C, we don’t observe a decreased dioxygen concentration and then the polymer degradation (FIG.4).
Question 3: Do our enzymes have the same enzymatic activity?
We tried to clone several laccases from T.thermophilus, E.coli, B. subtilis and Laccase 15 (from a uncultured bacterium) and several cytochromes C from S. oneidensis and Synechosystis sp.
We cloned with success two laccases (T. thermophilus and E.coli) and one cytochrome C (S.oneidensis). We tried to produce these proteins into the E.coli BL21 strain.
We purified the E.coli laccase and performed enzymatic tests to determine if it has an activity. To do this, we used the ABTS (2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)), which is oxidized by laccases. This compound is colored in green when it is oxidized.
Unfortunately, we don’t observe activity with purified E.coli laccase. We know that copper binds laccases. So we incubate our laccase with copper and with no success.
1) We produced the E.coli laccase in aerobic condition. However, the E.coli laccase could have a bad folding in these conditions and then needs to be produced in anaerobic condition.
2) Usually, a laccase shows a blue color given by copper presence. Our purified E.coli laccase is not blue. Maybe the copper is absent. Ni-NTA column is known to remove metal from protein. We can hypothesis that the use of the Ni-NTA is not appropriate.
In perspective, we would like to perform the production of our E.coli laccase in anaerobic condition. Using another purification column such as an anion-exchange column or size exclusion, we hope to conserve copper center and enzymatic activity.