"
Team:Fatih Turkey/Results
From 2011.igem.org
For our project lab executions;
1. LALF based anti-LPS factor system has been worked expectedly
2.we have constructed and submitted 12 biobrick. Three devices working in two for B.subtilis one for E.coli work expectedly.
3. Our B.subtilis specific promoter with SacB- extracellur signal- has been seen working well.
4. We have synthesized Reflectin 1a protein in E.coli; however, because we did not have enough time to purify it to visualize via aid-eye, we could not use Reflectin 1a device as biosensor for our lalf-LPS interaction in B.subtilis- E.coli mixed culture.
DISC EXPERIMENT1
5. There are b.subtilis with LALF in our B1 plate and B.subtilis without LALF in our B2 plate.For working of our LALF protein ,count of e.coli with RFP in B1 is lower than B2.
6. We spread fresh E.coli which produce RFP and put them onto 100uL Bacillus Subtilis supernatant which include LALF (plate D1) and no LALF protein producing Bacillus Subtilis supernatant(plateD2).As you can see where Bacillus supernatant with LALF exist ,there wasn’t any E.coli with RFP.
7. We spread fresh E.coli which produce RFP and put them onto 100uL Bacillus Subtilis which include LALF (plateC1 ) and no LALF protein producing Bacillus Subtilis (plateC2).As you can see where Bacillus with LALF exist ,there wasn’t any E.coli with RFP.
8. We saw that size of colonies with LALF protein is smaller than size of colonies without LALF protein and also antibiotic didn’t changed the results.
THE SUICIDE EXPERIMENT OF E. COLI
9. IPTG was caused of suicide of E.coli.Our promoter is IPTG inducable.So when we added IPTG in the media LALF synthezised.Result of this experiment we can say that E.coli kills itself.
For Human Practice; We have performed the activities as follows;
1. Providing Sporocide Method for prevention from spores of B.subtilis in further B.subtilis synthetic biology project.
2. Preparing a novel board game `Canvas Time` to inform people from any distinct education level about features of our project, laboratory equipments and interesting Biobrick of iGEM competition. .
3.Performing open-lab to public meetings such as gifted children studying in several schools of Ankara, undergraduate students from Hacettepe University Medical School and postgraduate students from Ankara University Medical School
4.Kindergarten synthetic biology trip to teach synthetic biology to five-year-old children.
RESULT: LALF find in the DNA sequence and mRNA sequence. We produced LALF protein by E. coli.
J04500 GFP LALF /pSB1AK3
Producing of expected part’s mRNA is confirmed by electrophoresis and sequence analysis.
As a result, mRNA of LALF protein has been produced, the promoter is producing both LALF and GFP sequences.
CONFIRMATION
After cloning procedure, we loaded our parts into gel to perform gel electrophoresis. As a result, we confirmed someof our parts by comparing destination of their bands in the gel with their prospected size.
In conclusion, BBa_K541915, BBa_K541545, BBa_K541515, BBa_K541596, BBa_K541800, BBa_K541501, BBa_K541502, BBa_K541503, BBa_K541504, BBa_K541505, BBa_K541506, BBa_K541516, BBa_K541526, BBa_K541546 are confirmed.
Their electrophoresis images are added below. Indicated places are confirmed gene sequences.
RESULT: LALF find in the DNA sequence and mRNA sequence. We produced LALF protein by E. coli.
J04500 GFP LALF /pSB1AK3
Producing of expected part’s mRNA is confirmed by electrophoresis and sequence analysis.
As a result, mRNA of LALF protein has been produced, the promoter is producing both LALF and GFP sequences.
DISC EXPERIMENT
There are b.subtilis with LALF in our B1 plate and b.subtilis without LALF in our B2 plate.For working of our LALF protein ,count of e.coli with RFP in B1 is lower than B2.
We spread fresh E.coli which produce RFP and put them onto 100uL Bacillus Subtilis supernatant which include LALF (plate D1) and no LALF protein producing Bacillus Subtilis supernatant(plateD2).As you can see where Bacillus supernatant with LALF exist ,there wasn’t any E.coli with RFP.
We spread fresh E.coli which produce RFP and put them onto 100uL Bacillus Subtilis which include LALF (plateC1 ) and no LALF protein producing Bacillus Subtilis (plateC2).As you can see where Bacillus with LALF exist ,there wasn’t any E.coli with RFP.
We saw that size of colonies with LALF protein is smaller than size of colonies without LALF protein and also antibiotic didn’t changed the results.
THE SUICIDE EXPERIMENT OF E. COLI
IPTG was caused of suicide of E.coli.Our promoter is IPTG inducable.So when we added IPTG in the media LALF synthezised.Result of this experiment we can say that E.coli kills itself.
After adding the FR solutions on all of plates, we measured their OD values in the end of this experiment we measured again and saw that the 1st, 2nd, 3rd, 4th plates OD values didn’t change. But the the OD value of 5th plate increased. 5th plate is the control group.
OFR is more effective than DFR for B,subtilis.
DFR is more effective than OFR for E.coli.
Sponsors
