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From 2013.igem.org

Contents 1 Experiment Results / Discussion 1.1 -The Experiments about EpCAM – Anti EpCAM Binding 1.1.1 Immunofluorescence c215 1.1.2 Immunofluorescence OmpA 2 Ellman’s assay experiment 3 Protein G binding experiment 4 Signal Peptide 5 DISCUSSION 5.1 Optimization of constitutive promoter for protein over expression 6 Discussion

Experiment Results / Discussion

-The Experiments about EpCAM – Anti EpCAM Binding

Firstly we wanted to test that can our bacteria produce C215 (Anti-EpCAM). To test this, we did Immunofluorescence experiments. We put OmpA (a signal peptide which send proteins to membrane) signal peptide sequence and his-tag sequence before C215 protein sequence in our one part. And then we did immunofluorescence and we waited to see a red circle around our bacteria and we saw. We added his-tag because C215 was staining thank to his-tag. During the experiments we used anti-his-tag, after than the substance which will paint… At this step we understood that our bacteria can produce C215 because we saw red circles. The second, we did another immunofluorescence experiment too. This experiment’s purpose was to understand that will EpCAM and our C215 bind? Because our experiment which the other immunofluorescence (the OmpA was using there) experiment, we understood that bacteria is producing C215, but we couldn’t understand anything about binding. This time we used cancer cells and we didn’t use bacteria, we used purified C215 to take some information about binding. We put our purified proteins in well-plates with cancer cells. We were expecting to see red color circle around cancer cells and to seeing cancer cells’ nucleus blue.

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Immunofluorescence c215

In our project we need that our anti-EpCAM active domain c215 bind the membrane of cancer cells. So we decided to make immunofluorescence experiment for tested that our c215 is binding or not? As you see in Pic. 1 the little red shining cells are our results and show that our c215 is working. With this results we saw that our nanofactory will probably bind to our cancer cells successfully.

Immunofluorescence OmpA

Before we start to made experiments we decided to use an alternative methods for detection cancer cells (Nano factory). We decided to use our bacteria as a nanofactory (quorum sensing). We added OmpA in front of our c215 so our bacteria can bind cancer cells. So we tested OmpA that produced by our bacteria and we made immunofluorescence experiments. So as a results see in Pic 2 (little red shining bacteria) our OmpA presented by our cells to outer membrane successfully. So that means we will use our bacteria as a Nanofactory.

Ellman’s assay experiment

It’s possible to measure the autoinducer concentration by ellman’s assay. Ellmans assay needs liquid culture of enzyme expressing bacteria so we preapare liquid cultures (with antibiotics) of them. After incubating liquid cultures add 0,1 mM substrate (SAH in 10X PBS containing %1 BSA) and incubated SAH added liquid cultures at room temprature for two hour. While the bacteria incubating, enzymes producted autoinducer 2. After incubating, santrifujed and measure the supernatants. We measure autoinducer concentration, enzymes production. We use transformed bacteria as a negative control. Ellman’s assay result:

This result telling us enzymes are expressed by e.coli and succesfully produced autoinducer2. Inducible promoter experiment To observe inducible promoters response, we co incubated two different bacteria. The first bacteria culture is expressing enzyme system and the second culture is including inducible promoter. At the experiment, we incubated them seperately for 16 hours and mixed them into one flask and 4 hour later, we add 0,1 mM SAH (with 10x PBS containing %1 BSA). Incubate with SAH for one day and, took 2 ml of liquid culture to santrifuge tube.Centrifuged it. We saw the red color at the pellet as you see below.

So, it means the enzyme system is working and inducible promoter is succesfully induced from AI-2. Discussion of ellman’s assay

 Our results clearly shows that we have overcome our challenges. According to ellman’s assay experience, our enzyme system is succesfully producing auto inducer 2. As described at design part, this molecule will induce bacteria for react to cancer cell.

We were searching for inducible system that, getting activated with cancer cell antigen precense. As an activating molecule, autoinducer2 activates LsrR-LsrK promoter system for express the killer protein by bacteria. Our co-incubation liquid cultures’ santrifuged tubes are—kırmızı görünüyor  we added rfp gene to continue of inducible promotor gene and rfp is marker of activating. So this co incubation result significantly shows us promoter system is activating in the precense of autoindcer2.

Protein G binding experiment

Our nanofactory includes antibody, Protein G and enzyme complex. We needed connection enzyme system between antibody as a linker.Herewith we used protein G which derived from group C and G Streptococcus strains and binds to the Fc region of numerous immunogloblulins. Protein G facilitates binding of enzyme construct to the Fc region of the targeting antibody.Also we was cloned into pSB1C3 and we express this protein in BL21 strain of E. coli. Futhermore we tagged his6x at N terminus of protein G for purificaiton.

The aim of protein G and Ig G binding experiment is to make a complex with two parts of nanofactory and construct our nanofactory.

 Our nanofactory includes antibody, Protein G and enzyme complex. There is natural affinity of protein G to bind to antibody in phosphate buffer. So we incubated protein G and antibodies in 10mM phosphate buffer for 2,5 hours then we measure the quantity of fused proteins via Western Blotting experiment.

Result;

Our protein G weight is 25 kDas and our combined Protein G + antibody protein’s weight is about 50 kDas. We accepted the experiment result that protein G nearby of 25 kDas line and the complex nearby of 50 kDas. The experiment throughput was the same as we accepted as you see above. The result show that we have produced nanofactory for definite.

Signal Peptide

A signal peptide (sometimes referred to as signal sequence, leader sequence or leader peptide) is a short (5-30 amino acids long) peptidepresent at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.[1] In prokaryotes, signal peptides direct the newly synthesized protein to the SecYEG protein-conducting channel, which is present in the plasma membrane. 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.

DISCUSSION

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. Also we get another fine results from our GFP-HlyA that we found high amount of GFP in our samples more than our Negative(-) Control. Another good results that our Positive Control which is GFP that added in LB showed maximum amount of GFP in graphics. So we can said that our controls were working too.

• Tang et al 2008 ., Jin-bao Tanga, Hong-ming Yanga, Shu-liang Songb, Peng Zhub, Ai-guo Jib

(Effect of Glycine and Triton X-100 on secretion and expression of ZZ–EGFP fusion protein) 1)Blobel G, Dobberstein B. (Dec. 1975). "Transfer of proteins across membranes. I. Presence of proteolytically processed and unprocessed nascent immunoglobulin light chains on membrane-bound ribosomes of murine myeloma."

Optimization of constitutive promoter for protein over expression

Over expression experiment’s aim is over synthesis of protein in a new medium that we prepared. We thought that’s a good idea over expressing of protein by the bacteria to observe the net results. So we decided to prepare the “best medium” for the bacteria. Then we generated different media combinations. We generated the combinations by altering conditions of bacteria, liquid culture incubation time, bacteria strain and temperature of incubator. So at the end of the experiment there were 60 different conditions and media we designed. Different media and conditions in our experiment:

After preparing the media we started the experiment stage. Our experiment includes protein isolation and SDS page parts. We got the total amount of protein of the bacteria after the protein isolation by using sonicator. Then we measure the band of our proteins in the SDS Page experiment. Finally we chose the winner a medium called TNY, BL21, 24H, 37C SDS Page results;

           TNY, BL21, 37C, 24Hr                        TNY, NEB10, 37C, 24Hr

Discussion

We were excepting the LB for the winner of the experiment but we surprised by seeing the result. Because the medium that prepared by us and called TNY was the best. The content of TNY and LB is the similar but the component percentage of media is different. It is very interesting that low rated medium is better than the LB. There is small percentage dissimilarity between the media like 7/200 and 4/200. Our TNY is preparing by diluting 4 gr TNY extract with distilled water. We observed the protein expression level of TNY 10 times more than LB as you see above.