BBa_K3309001 as improvement over BBa_K1122674
An induction expression test using IPTG or under the control of the Pan promoter, of the fusion protein adhB-pdc from BBa_K1122673 was performed in E. Coli S17.1, using the plasmid pBBR1MCS-2 as the expression vector.
An SDS-page gel of this expression test (Gel SDS - Page 2) showed that the production of this fusion protein is performed in quite large quantities as early as 2 hours after induction at IPTG and increases further until 20 hours after induction at S17.1.
Similarly, the production of this protein was tested after placing it under the control of the Pan promoter, which is a strong and constitutive promoter.
Indeed, the results on the SDS-Page gel show that under control of this promoter, the quantity of protein produced seems comparable to that observed after 20 hours of induction when it is controlled by the inducible promoter Lac.
These results then show that this promoter allows a strong expression of the protein in a constitutive way.
In addition, it has been shown that this promoter is inducible to the presence of certain heavy metals in C. Metallidurans such as Cobalt Co(II), Cadmium Cd(II), Manganese Mn(II), Zinc Zn(II), Copper Cu(II) or Nickel Ni(II).
These elements may suggest that this construction would therefore allow even better protein production when C. Metallidurans is exposed to these heavy metals, particularly in our case, Cadmium.
Gel SDS - Page 2 : 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.
References
A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans. - PubMed - NCBI. https://www.ncbi.nlm.nih.gov/pubmed/20517979.