Team:Fatih Turkey/Results

From 2011.igem.org

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<p><em>1. </em><em>technique;</em> in case of applying on surfaces, LALF protein can work like a new sterilization material like gluteraldehyte or alcohol that are commonly used for hospital and lab sterilization.<em> </em></p>
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<p><em>2. </em><em>Alternative cure for septic shock;</em> after gram negative bacterial infections; LPS, which is the main component of cell wall of gram negative bacteria, can cause deathly conditions that is generally called “septic shock”. In our project, our LALF protein binds this material strictly, thus LALF can be used as an alternative cure method for such condition.<em> </em></p>
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<p><em>3. </em><em>Possible new structure of antibiotic-like drug; </em>antibiotics commonly effect on production of cell wall of bacteria. This is done by inhibiting the source of production. On the other hand, our LALF protein directly binds the cell wall material and stops the bacteria growth. Thus, a new antibiotic like drug made of LALF may be produced and used in some infections.<em> </em></p>
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<p><em>4. </em><em>Possibility of producing longtime sterile layers; </em>if any kind of object is produced by covering with LALF, that object can stay sterile for long time because of gram negative bacteria protection of LALF. In case of a possible infection on surface, LALF will stop bacteria growth.<em> </em></p>
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<p><em>5. </em><em>Alternative cure method for bacterial diseases; </em>diseases like urinary system infection or gastritis are caused by gram negative bacteria.<em> </em>In the course of applying our bacteria that produce LALF in the infectious cite, infection of harmful bacteria can be prevented or stopped. This may suggest an alternative cure for such diseases.<em> </em></p>
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<p>On the other hand, our secondary protein, reflectin, can be used in varying applications.</p>
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<p><em>1. </em><em>Camouflage material; </em>this protein allows its host, cephalopod, to hide when cephalopod hunts or to defend itself from its hunters. In the course of development of reflectin protein, a new camouflage material may be produced for humans.<em> </em></p>
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<p><em>2. </em><em>New material of dye for varying applications; </em>Dye that changes its color can be used almost every colorful application. From fireworks to wall paints, from windows of buildings to road signs, this ability can be very useful.<em></em></p>
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<p><em>3. </em><em>Possibility of producing specialized light colors; </em>in some circumstances, a unique light color may be necessary in order to gain maximum output like in greenhouses or dark rooms in photography industry. Reflectin reflects the light by changing its wavelength; therefore the light that is reflected has only one color. This ability can be applied in such application that mentioned above.<em></em></p>
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<p><div>
 
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<p><em></em><em>For our project lab executions;</em></p>
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</p><p><strong><em></em><em>For our project lab executions;</em></strong></p>
<p> 1. LALF based anti-LPS factor system has been worked expectedly<em> </em></p>
<p> 1. LALF based anti-LPS factor system has been worked expectedly<em> </em></p>
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B.subtilis one for E.coli work expectedly.
B.subtilis one for E.coli work expectedly.
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<em> </em></p>
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<em> </em></em></p><em>
<p><em>3. Our B.subtilis specific promoter with SacB- extracellur signal- has been seen  
<p><em>3. Our B.subtilis specific promoter with SacB- extracellur signal- has been seen  
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working well.<em> </em></p>
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working well.<em> </em></em></p><em>
<p><em>4. We have synthesized Reflectin 1a protein in E.coli; however, because we did not  
<p><em>4. We have synthesized Reflectin 1a protein in E.coli; however, because we did not  
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Reflectin 1a device as biosensor for our lalf-LPS interaction in B.subtilis-
Reflectin 1a device as biosensor for our lalf-LPS interaction in B.subtilis-
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E.coli mixed culture. <em> </em></p>
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E.coli mixed culture. <em> </em></em></p><em>
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<p>DISC EXPERIMENT1</p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>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.</p>
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<p>THE SUICIDE EXPERIMENT OF E. COLI</p>
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<p>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.</p>
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<p><em></em><em>For Human Practice; We have performed the activities as follows;</em></p>
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<p><strong><em></em><em>For Human Practice; We have performed the activities as follows;</em></strong></p>
<p>1. Providing Sporocide Method for prevention from spores of B.subtilis in further  
<p>1. Providing Sporocide Method for prevention from spores of B.subtilis in further  
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B.subtilis synthetic biology project.<em> </em></p>
B.subtilis synthetic biology project.<em> </em></p>
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<p>2. On the other hand, our secondary protein, reflectin, can be used in varying applications.</p>
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<p>2. Preparing a novel board game `Canvas Time` to inform people from any distinct  
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<p>3. Preparing a novel board game `Canvas Time` to inform people from any distinct  
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education level about features of our project, laboratory equipments and  
education level about features of our project, laboratory equipments and  
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interesting Biobrick of iGEM competition.
interesting Biobrick of iGEM competition.
.</p>
.</p>
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<p>4.Performing open-lab to public meetings such as gifted children studying in  
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<p>3.Performing open-lab to public meetings such as gifted children studying in  
several schools of Ankara, undergraduate students from Hacettepe University  
several schools of Ankara, undergraduate students from Hacettepe University  
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Medical School and postgraduate students from Ankara University Medical School
Medical School and postgraduate students from Ankara University Medical School
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<p>5.Kindergarten synthetic biology trip to teach synthetic biology to five-year-old  
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<p>4.Kindergarten synthetic biology trip to teach synthetic biology to five-year-old  
children.  
children.  
  </p>
  </p>
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<p><strong><br />
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<p><strong><br>
</strong></p>
</strong></p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/a/af/S3.png"></p>
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<p>&nbsp;</p>
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<p><span style="text-decoration: underline;">RESULT</span>: LALF find in the DNA sequence and mRNA sequence. We produced LALF protein by E. coli.</p>
 +
<p>&nbsp;</p>
 +
<p>J04500 GFP LALF /pSB1AK3</p>
 +
<p>&nbsp;</p>
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<p>Producing of expected part’s mRNA is confirmed by electrophoresis and sequence analysis.</p>
 +
<p>As a result, mRNA of LALF protein has been produced, the promoter is producing both LALF and GFP sequences.</p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/d/d1/Sss1.png"></p>
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<p>&nbsp;</p>
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<p>CONFIRMATION</p>
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<p>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.</p>
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<p>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.</p>
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<p>Their electrophoresis images are added below. Indicated places are confirmed gene sequences.</p>
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<p>&nbsp;</p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/d/db/M1.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/c/cd/M2.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/7/77/M3.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/5/5d/M4.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/4/49/M5.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/6/6c/M6.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/6/6c/M7.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/0/07/M8.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/c/c0/Ms4.png"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/6/61/M10.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/4/48/M11.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/a/aa/M12.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/2/25/M13.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/a/a0/Ms3.png"></p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/a/a5/M16.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/6/64/Ms2.png"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/6/6c/Ms1.png"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/3/30/M19.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/5/5c/M20.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/d/d9/M21.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/3/3b/M22.jpg"></p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/8/85/M23.jpg"></p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/a/af/S3.png"></p>
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<p>&nbsp;</p>
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<p><span style="text-decoration: underline;">RESULT</span>: LALF find in the DNA sequence and mRNA sequence. We produced LALF protein by E. coli.</p>
 +
<p>&nbsp;</p>
 +
<p>J04500 GFP LALF /pSB1AK3</p>
 +
<p>&nbsp;</p>
 +
<p>Producing of expected part’s mRNA is confirmed by electrophoresis and sequence analysis.</p>
 +
<p>As a result, mRNA of LALF protein has been produced, the promoter is producing both LALF and GFP sequences.</p>
 +
<p><img src="https://static.igem.org/mediawiki/igem.org/d/d1/Sss1.png"></p>
 +
<p>&nbsp;</p>
 +
<p>DISC EXPERIMENT</p>
 +
<p><img src="https://static.igem.org/mediawiki/2011/d/d1/Sss1.png"></p>
 +
<p>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.</p>
 +
<p><img src="https://static.igem.org/mediawiki/igem.org/f/fd/Sss2.png"></p>
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<p>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.</p>
 +
<p><img src="https://static.igem.org/mediawiki/2011/8/85/Sss3.png"></p>
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<p>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.</p>
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<p>&nbsp;</p>
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<p>&nbsp;</p>
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<p><img src="https://static.igem.org/mediawiki/2011/f/f9/IMG_0331.JPG"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/1/1f/IMG_0335.JPG"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/a/a6/IMG_0339.JPG"></p>
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<p>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.</p>
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<h2>THE SUICIDE EXPERIMENT OF E. COLI</h2>
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<p>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.</p>
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<p><img src="https://static.igem.org/mediawiki/igem.org/6/6e/S1.png"></p>
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<p><img src="https://static.igem.org/mediawiki/2011/4/40/S2.png"></p>
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<p>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 1<sup>st</sup>, 2<sup>nd</sup>, 3<sup>rd</sup>, 4<sup>th</sup> plates OD values didn’t change. But the the OD value of 5<sup>th</sup> plate increased. 5<sup>th</sup> plate is the control group.</p>
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<p><img src="https://static.igem.org/mediawiki/2011/0/02/DSC03586_-_Kopya.JPG"></p>
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<p>&nbsp;</p>
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<p>OFR is more effective than DFR for B,subtilis.</p>
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<p>DFR is more effective than OFR for E.coli.</p>
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<p><div>
 
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</em></em></em></div>
    </div>
    </div>
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       </div>

Latest revision as of 08:22, 5 October 2011

deneme baslik

Project

Results

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.

2011 © Fatih Medical School

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