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OmpA codes for transport of the intercellular produced protein over the outer membrane by the formation of a  bèta-barrel. However, to transport over the outer membrane, first the protein must be transported over the inner membrane. For this,  the OmpA contains a sequence, called the Sec-peptide,  which codes forthis transport over the inner membrane. The Sec-peptide binds to a transporter on the inner membrane which allows transport to the periplasm. Subsequently, the OmpA folds into the outer membrane due its tertiary structure: formation of the bèta-barrel . Mfp5-GFP-LVA then folds through the barrel to the outside. In order to confirm that this transport system is working properly, we needed to have a negative control. Therefore we designed two ways of removing the coding sequence of the Sec-peptide: site-directed mutagenesis and a combination of PCRs, digestion and ligation. In site-directed mutagenesis we introduced two stop codons in the coding sequence of the Sec-peptide to prevent this part from being transcribed and translated. A start codon was present shortly after the location of the newly introduced stop codons. Since the sequence is very close to the promotor translation of the parts afterwards, this sequence should still function properly. This is not certain however, and so we had a more elaborate second approach. This involved using PCR to get the coding sequence before and, separately, after the Sec-peptide. Paying attention to the reading frame, we digest and ligate these sequences to have once again  a coding sequence analogous to the original one, but without the Sec-peptide sequence. This led to a construct which contained in sequential order the p-BAD promotor, Mfp-5, GFP and  OmpA.

Transport construct.jpg

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