An expression test by induction with IPTG or under the control of the Pan promoter, the adhB-pdc fusion protein from BBa_K1122673 part was performed in E. Coli S17.1, using the plasmid pBBR1MCS-2 as an expression vector.
An SDS-page gel of this expression test showed that the fusion protein was produced in relatively large quantities as early as 2 hours after induction at IPTG.
Based on the most reliable theoretical ORF (found whith ORF Finder), it is possible to estimate the molecular weight of this protein (found with Isoelectric) at 100.92 kDa.
These results were already expected by the 2013 Edinburgh team, but they obtained a protein of about 80 kDa.
The protein obtained here has a size of about 100 kDa. This size, under the control of the Lac promotor in pBBR1MCS-2.
The ORF produced by the bacterium is therefore possibly larger than expected if it contains a fragment of the LacZα gene.
Despite this, the protein obtained has a molecular weight very close to that expected, which gives an indication of its good production in a complete way.
Other elements could be observed during the expression tests of this protein, both in E. Coli S17.1 and in C. metallidurans CH34 or ΔpbrR.
First, bacterial growth tests show that strong expression of this protein induces a significant decrease in growth, compared to the wild type or non-expressing strain of this protein.
The large size of this protein and its strong expression (both by induction at IPTG and under the control of the Pan promoter) may explain the mobilization of additional energy for protein production, which is no longer used for growth and bacterial division.
Secondly, the results of the DNS assay (see results) of reducing sugars do not show any difference in the consumption of these sugars (mainly glucose) between strains expressing or not expressing the fusion protein in C. metallidurans during its growth.
Indeed, the use of pyruvate by this fusion protein is not the majority compared to that of the bacterium's metabolism (respiration).
This shows that the use of pyruvate is not diverted mainly for ethanol production and therefore does not induce stress in terms of energy production (fermentation producing less energy than respiration) for the bacteria.
Expression test of the fusion protein adhB - pdc from BBa_K1122673 part (noted BB) by IPTG induction in E. Coli S17.1 and under the Pan promoter control (noted Pan) in C. metallidurans CH34 or on C. Metallidurans with pbrR gene deletion (noted ΔpbrR) in different culture conditions and with or without activation on 284 medium supplemented by lead and cadmium durring 15 minutes (noted activated), on SDS page gel.