Part:BBa_K1202115:Experience

Signal Peptide experiments, TorA

In this part of our project we tested are our signal peptides is working or not

To secrete GFP protein via signal peptide to extracellular membrane.

In overall project, our aim related with signal peptide is to secrete apoptotic proteins outer membrane. If this construct secrete GFP successfully, that means signal peptide works and TorA can be used in Apoptin or E4orf4 export.

And also we use constitutive promoter in our designs because we need high amount of protein and we want to our bacteria always produce our proteins.

We made santrifuge our signal peptides’ (TorA-GFP ve GFP-HlyA) liquid culture falcons and then get supernatant from falcons. Then we measured values of absorption of GFP in our supernatant in suitable wavelength. Results show that we found high amount of GFP in our supernatants.

Result;

Column scale(y) shows us quantity amount of GFP in the solution.

Firstly we made liquid culture in our TorA-GFP and GFP-HlyA experiments. We added TritonX half of our TorA-GFP samples and didn’t add any chemicals our other TorA-GFP samples . After the Overnight process we made santrifuge and we get supernatants from our samples because we wait our proteins in supernatant.

The results of the supernatant of the TorA-GFP samples that we didn’t add TritonX & Glycine were over 0. So we thought that we got this results becuse of the cellular necroses that caused secretion of low amount of GFP. Because of the secretion of the GFP in periplasmic gap we used %2 TritonX*+%1 Glycine* that wrote in article and we destroy outer memrane. We got this concentration from article.

Our results in our TorA-GFP experiments that our samples that added TritonX contains more GFP amount than Negative(-) Control. So this results showed us that our signal peptides worked properly and our chemicals TritonX and Glycine showed their effects

Tat Apoptin His

We controlled our genes in gel electrophoresis. Electrophoresis showed us the transformation of new genes was successful.There ise gel electrophoresis results below;

TAT Apoptin purification experiment ;

To purify the recombinant proteins, cells were spun down from 50 mL of culturesupernatant and resuspended in phosphate buffer saline.PBS contained PMSF in order to prevent proteis from proteases. The mixture was then sonicated on ice eight times for 30 second with a 52% pulsedactivity cycle (MISONIX SonicatorW 300). Next, the lysate was centrifuged for 30 min at 10,000 rpm to removethe cell debris. The resulting cell supernatant was loadedonto HIS select affinity column (Sİgma Aldrich)) for protein purification using the standard procedure.. The totalprotein concentration of each collected fraction fromthe column was determined using Bradford protein assay with bovine serum albumin actingas the reference protein. The purity of the protein fromeach fraction was analyzed by 12.5% SDS-PAGE andthen the resulting gels were Western blotted using monoclonal anti-HIS antibody.

Using western blot analysis, it was observed that; our proteins expressed and purified effectively.

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