Team:UPMC PARIS/Parts

Impact UPMC



Part submission : BBa_K2504000


Introduction

The 17 kilodalton protein called Skp is a molecular chaperone that resides in the periplasm of Escherichia coli. The SKP protein folding enables the expression of high-level recombinant proteins which contributes to the development of therapeutic molecules to treat misfolded proteins in some diseases. Drug development of such molecules can lead to production with bacteria of anti-cancer vaccines. Consequently, Skp has been shown to improve expression of various scFvs, in addition to larger Fab fragments and intact immunoglobulins (Raphael Levy and all,2011). This improvement of bacterial strain by genomic integration could brings a robust biological tool related to our project and an alternative in the use of antibiotics. Other genomic integrations will be realized in the continuity of our project to avoid maintaining plasmids through antibiotics, and to facilitate the use of our factory. In a second time it could be also relevant to permit production of others helper proteins like GroEL/ES.



Achievements

We submitted our biobrick sequence to the iGEM community, and we documented the experimental characterization of this part on the Main Page of that Part’s Registry entry.



Method


We started from the cytosolic form of Skp under the control of T7 promoter, ended by a T7 terminator and flanked by iGEM prefix and suffix by synthesized by IDT.

We used the NotI site to insert the sequence into the iGEM plasmid pSB1C3 and we transformed competent E. coli DH5α.

After transformation, we performed a plasmid DNA extraction. We selected recombinante clone by PCR using VF and VF2 primers. We collaborate with sequencing platform Eurofin in order to verify our DNA sequence by sequencing and dry the remaining DNA to send it to the iGEM foundation.


Results


Figure 6 Agarose gel electrophoresis (1.5%) after migration of PCR products for the verification of the insertion the biobrick BBa_K2504000



The presence of a band at 695pb suggest that the integration in the pSB1C3 worked as expected. We submit our biobrick with n=5 and sand a sample to sequencing to ensure the quality of our biobricks.

Thus our Biobrick encodes for a cytoplasmic form of the chaperones Seventeen kilodalton protein (Skp). The Biobrick construction is composed of a T7 promoter, the coding DNA sequence of Skp protein truncated by its localisation signal sequence and a T7 terminator.


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