Team:Fatih Turkey/Experiments
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<div class="meta-left-full"> | <div class="meta-left-full"> | ||
+ | <div style="font-size:15px; color: #333;"> | ||
+ | A)EXPERIMENTS | ||
+ | <p style="padding-left: 30px;">1)<a href="#1">Disc experiments 1</a></p> | ||
+ | <p style="padding-left: 30px;">2)<a href="#2">Disc experiments 2</a></p> | ||
+ | <p style="padding-left: 30px;">3)<a href="#3"> ?</a></p> | ||
+ | <p style="padding-left: 30px;">4)<a href="#4">THE SUICIDE EXPERIMENT OF E. COLI</a></p> | ||
+ | <p style="padding-left: 30px;">5)<a href="#5">THE EFFECT OF FENTON REAGENT SOLUTION ON THE BIOFILM</a></p> | ||
+ | <p style="padding-left: 30px;">6)<a href="#6">The Difference between Colonies Containing and Not Containing Gene Parts of Reflectin Protein</a></p> | ||
+ | <p style="padding-left: 30px;">7)<a href="#7">Experiment of Supernatant</a></p> | ||
+ | <p style="padding-left: 30px;">8)<a href="#8">Experiment of liquid culture</a></p> | ||
+ | B)<a href="#B">CONFIRMATION</a> | ||
+ | C)<a href="#C">CHARACTERIZATION</a> | ||
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- | <p><strong><span style="text-decoration: underline;"> | + | <p><strong><span style="text-decoration: underline;">Disc Experiment</span></strong></p> |
<p>We prepared an experiment like the disc experiments methods used to see antibiotic effectiveness, in order to see the effect of B. Subtillis with LALF on E. Coli.</p> | <p>We prepared an experiment like the disc experiments methods used to see antibiotic effectiveness, in order to see the effect of B. Subtillis with LALF on E. Coli.</p> | ||
<p>By adding E. Coli on Bacillus Subtilis;</p> | <p>By adding E. Coli on Bacillus Subtilis;</p> | ||
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<h1><em><span style="text-decoration: underline;"> </span></em></h1> | <h1><em><span style="text-decoration: underline;"> </span></em></h1> | ||
<h1><em><span style="text-decoration: underline;"> </span></em></h1> | <h1><em><span style="text-decoration: underline;"> </span></em></h1> | ||
- | < | + | |
+ | |||
+ | |||
+ | <br><hr><span id="3"> | ||
+ | <img src="https://static.igem.org/mediawiki/igem.org/d/db/M1.jpg" /></span><br> | ||
+ | 1: This part includes a gram positive promoter, an RBS sequence and SacB signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/c/cd/M2.jpg" /><br> | ||
+ | 2:This part includes a gram positive promoter, an RBS sequence and LipA signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/7/77/M3.jpg" /><br> | ||
+ | 3: This part includes a constitutive promoter, an RBS sequence and Tat signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/5/5d/M4.jpg" /><br> | ||
+ | 4:This part includes a IPTG inducible promoter, an RBS sequence and Tat signal peptide sequence to synthesize the protein outside of the cell. This image shows that indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/4/49/M5.jpg" /><br> | ||
+ | 5:Our “LALF (limulus anti-lipopolysaccharide factor)” protein allows stopping any kind of gram negative bacteria growth by binding their cell wall material, LPS. This image shows that indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/6/6c/M6.jpg" /><br> | ||
+ | 6:Our “reflectin” protein has the ability to reflect the light by changing its wavelength, thus its color. In our project, we planned to use this protein as an indicator on E.coli whether our LALF protein works properly or not. This image shows that indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/6/6c/M7.jpg" /><br> | ||
+ | 8:This part is a well-studied gram positive promoter. We planned to use this gene in the case of our signal peptide sequences do not work. When the protein, which is attached to this gene, is produced, we planned to blow up the bacteria; therefore synthesized protein would be in supernatant. This image shows that indicated part is considered as confirmed. | ||
+ | |||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/igem.org/0/07/M8.jpg" /><br> | ||
+ | 500: To perform cloning perfectly, we used pSB1C3 in E.coli. This vector is also the main vector for our parts that works only in E.coli. All of our parts are also inserted into this vector; because it is declared that all parts must be sent to Registry in pSB1C3. This image shows that this part is confirmed. | ||
+ | |||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/c/c0/Ms4.png" /><br> | ||
+ | 501: To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes a gram positive promoter, RBS sequence and SacB signal peptide sequence. In the image, it can be seen that this part is confirmed. | ||
+ | |||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/6/61/M10.jpg" /><br> | ||
+ | 502: To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes a gram positive promoter, RBS sequence and LipA signal peptide sequence. In the image, it can be seen that this part is confirmed. | ||
+ | |||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/4/48/M11.jpg" /><br> | ||
+ | 505:To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes the sequence of our LALF protein. In the image, it can be seen that this part is confirmed. | ||
+ | |||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/a/aa/M12.jpg" /><br> | ||
+ | 506:To perform ligation procedure appropriately, we designed an alternative part for all of our material parts. We inserted our promoters and proteins into pSB1C3 in order to have the alternative part that possesses different antibiotic resistance, comparing to original vectors of the parts. Moreover, it is wanted that all parts must be sent to Registry in pSB1C3. This part includes the sequence of our reflectin protein. In the image, it can be seen that this part is confirmed. | ||
+ | |||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/2/25/M13.jpg" /><br> | ||
+ | 800: In our project, we planned to use gram positive bacteria to synthesize our LALF protein in order to stop gram negative growth. On the other hand, to identify effectively whether our protein works or not, we decided to use reflectinprotein and to produce it from both of our bacteria; B.subtilis and E.coli. Thus, we determined to use a shuttle vector that works both bacteria kinds. This vector has ampicillin resistance for E.coli and chloramphenicol resistance for B.subtilis. In the image, it can be seen that this part is confirmed. | ||
+ | |||
+ | <hr> | ||
+ | <img src="https://static.igem.org/mediawiki/2011/a/a0/Ms3.png" /><br> | ||
+ | 900:In our project, we planned to use gram positive bacteria to synthesize our LALF protein in order to stop gram negative growth. On the other hand, to identify effectively whether our protein works or not, we decided to use reflectin protein and to produce it from both of our bacteria; B.subtilis and E.coli. Thus, we determined to use a shuttle vector that works both bacteria kinds. This vector has chloramphenicol resistance for both of bacteria kinds. This part also includes an RFP sequence. In the image, it can be seen that this part is confirmed. | ||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/igem.org/a/a5/M16.jpg" /><br> | ||
+ | 545: This part is designed; because it is wanted that all parts must be sent to headquarters in pSB1C3 vector. It possesses a promoter that works in gram negative bacteria, Tat signal sequence in order to synthesize the protein outside and LALF protein gene. In this image, indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2011/6/64/Ms2.png" /><br> | ||
+ | 815: In order to stop gram negative bacteria growth, our LALF protein must be synthesized by another type of bacteria, gram positive. Thus, we designed a special gene that works in only gram positive bacteria by inserting a promoter specialized for gram positive. Also, SacB signal sequence that allows us synthesizing the protein outside and LALF protein gene is included.All components are inserted into 800 that isdesigned for gram positive. In this image, indicated part is considered as confirmed | ||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2011/6/6c/Ms1.png" /><br> | ||
+ | 915: In order to stop gram negative bacteria growth, our LALF protein must be synthesized by another type of bacteria, gram positive. Thus, we designed a special gene that works in only gram positive bacteria by inserting a promoter specialized for gram positive. Also, SacB signal sequence that allows us synthesizing the protein outside and LALF protein gene is included.All components are inserted into 900 that is designed for gram positive. In this image, indicated part is considered as confirmed. | ||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2011/5/54/M18.jpg" /><br> | ||
+ | 925:In order to stop gram negative bacteria growth, our LALF protein must be synthesized by another type of bacteria, gram positive. Thus, we designed a special gene that works in only gram positive bacteria by inserting a promoter specialized for gram positive. Also, LipA signal sequence that allows us synthesizing the protein outside and LALF protein gene is included.All components are inserted into 900 that is designed for gram positive. In this image, indicated part is considered as confirmed. | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2011/3/30/M19.jpg" /><br> | ||
+ | 516: This part is designed; because all parts must be sent to headquarters in pSB1C3 vector. In this image, indicated part is considered as confirmed. | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2011/5/5c/M20.jpg" /><br> | ||
+ | 526: This part is designed; because all parts must be sent to headquarters in pSB1C3 vector.In this image, indicated part is considered as confirmed. | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2011/d/d9/M21.jpg" /><br> | ||
+ | 536:In our experiments, we aimed to indicate whether our LALF protein works or not, we used an alternative indicator protein, reflectin. This part is designed to work in gram negative E.coli. A constitutive promoter, a Tat signal sequence that allows synthesizing the protein outside and reflectin protein gene is included in pSB1C3. In this image, indicated part is considered as confirmed. | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2011/3/3b/M22.jpg" /><br> | ||
+ | 546:In our experiments, we aimed to indicate whether our LALF protein works or not, we used an alternative indicator protein, reflectin. This part is designed to work in gram negative E.coli. An IPTG inducible promoter, a Tat signal sequence that allows synthesizing the protein outside and reflectin protein gene is included in pSB1C3. In this image, indicated part is considered as confirmed. | ||
+ | |||
+ | <hr> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/igem.org/8/85/M23.jpg" /><br> | ||
+ | 596:In our experiments, we aimed to indicate whether our LALF protein works or not, we used an alternative indicator protein, reflectin. We designed an alternative part in the case that our signal sequences do not work. This part includes a promoter that works in only gram negative and reflectin protein gene. All components are inserted into pSB1C3. In this image, indicated part is considered as confirmed. | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h1><span style="text-decoration: underline;" id="4">THE SUICIDE EXPERIMENT OF E. COLI</span></h1> | ||
<p><em>Our K541545 part contains IPTG promoter and the gene part of LALF protein on a backbone which has resistance to chloramphenicol. We transported this part to E. coli for testing whether E. coli would kill itself or not.</em></p> | <p><em>Our K541545 part contains IPTG promoter and the gene part of LALF protein on a backbone which has resistance to chloramphenicol. We transported this part to E. coli for testing whether E. coli would kill itself or not.</em></p> | ||
<p><strong>Experiments of plates with and without IPTG<em> </em></strong></p> | <p><strong>Experiments of plates with and without IPTG<em> </em></strong></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong> </strong></p> | <p><strong> </strong></p> | ||
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- | <p><strong><span style="text-decoration: underline;" | + | <p><strong><span style="text-decoration: underline;" id="5">THE EFFECT OF FENTON REAGENT SOLUTION ON THE BIOFILM</span></strong></p> |
- | + | ||
<p>Fenton Reagent solution is a solution that can kill the B. Subtilis spors and bacteria. It kills the spors by oxidizing with its Cu ions.</p> | <p>Fenton Reagent solution is a solution that can kill the B. Subtilis spors and bacteria. It kills the spors by oxidizing with its Cu ions.</p> | ||
<p>We prepared this solution in two ways one with the hydrogen peroxide (OFR) according to the literature and the other with distilized water (DFR). In our experiments we learned which of these two solutions is more effective on E. Coli, B. Subtilis or the the spores of B. Subtilis and degree of its effectiveness.</p> | <p>We prepared this solution in two ways one with the hydrogen peroxide (OFR) according to the literature and the other with distilized water (DFR). In our experiments we learned which of these two solutions is more effective on E. Coli, B. Subtilis or the the spores of B. Subtilis and degree of its effectiveness.</p> | ||
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<p><img src="https://static.igem.org/mediawiki/2011/4/40/S2.png"></p> | <p><img src="https://static.igem.org/mediawiki/2011/4/40/S2.png"></p> | ||
<p><img src="https://static.igem.org/mediawiki/2011/a/af/S3.png"></p> | <p><img src="https://static.igem.org/mediawiki/2011/a/af/S3.png"></p> | ||
- | <p><strong><span style="text-decoration: underline;">The Difference between Colonies Containing and Not Containing Gene Parts of Reflectin Protein</span></strong></p> | + | |
+ | |||
+ | <p><strong><span style="text-decoration: underline;" id="6"><h1>The Difference between Colonies Containing and Not Containing Gene Parts of Reflectin Protein</h1></span></strong></p> | ||
<p>We transfered the plasmid which includes the gene part of reflectin protein (j04450 shuttle, j04500 backbone, and 1006) to E. Coli and we saw that there is a difference between reflectin including colonies and the colonies which does not include reflectin protein.</p> | <p>We transfered the plasmid which includes the gene part of reflectin protein (j04450 shuttle, j04500 backbone, and 1006) to E. Coli and we saw that there is a difference between reflectin including colonies and the colonies which does not include reflectin protein.</p> | ||
<p>We saw that the plate which does not include reflectin protein is normally transparent. But the other plate which includes reflectin protein seemed white. And this is showing us that our plasmid is working and the protein is produced by E. coli.<br /> | <p>We saw that the plate which does not include reflectin protein is normally transparent. But the other plate which includes reflectin protein seemed white. And this is showing us that our plasmid is working and the protein is produced by E. coli.<br /> | ||
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<p>Consequently, it’s confirmed that the 1006 gene part is producing the reflectin protein</p> | <p>Consequently, it’s confirmed that the 1006 gene part is producing the reflectin protein</p> | ||
<p> </p> | <p> </p> | ||
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+ | <div> | ||
+ | <p><h1><span id="7">Experiment of Supernatant</span></h1></p> | ||
+ | </div> | ||
+ | <p>We wanted to investigate to effects of supernatant includes LALF protein to E.coli in this experiment.</p> | ||
+ | <p>Therefore we put 8 ul LB broth into falcons.then we added them E.coli which has rfp and incubated for 12 hours in 37⁰C .We put pure E.coli into only one falcon for control.We added 1ul,10ul and 100ul supernatant with LALF and without LALF.</p> | ||
+ | <p>We diluated them 7 times 2-4-8-12hours later then we spreaded 10ul to plates which has 1/0,5 l Cloramphenicol.We did santrifuge liquid culture which incubated for 12 hours at 37⁰C at 7000 rpm for 5 minutes because of preparing supernant with LALF and without LALF.Then we passed them from the 0,2 filter.</p> | ||
+ | <p>Plates were spreaded after 2 hours later:</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/5/5f/2saat.jpg"></p> | ||
+ | <p>1a)We added 1ul supernatant includes LALF into left plate and 1ul supernatant without LALF into right plate</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/1/12/2saat_b.jpg"></p> | ||
+ | <p>1b)We added 10 ul supernatant includes LALF into left plate and 10 ul supernatant without LALF into right plate</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/d/db/2_saat_c.jpg"></p> | ||
+ | <p>1c)We added 100ul supernatant includes LALF into left plate and 100ul supernatant without LALF into right plate</p> | ||
+ | <p>Plates were spreaded after 4 hours later;</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/9/9d/4saat_a.jpg"></p> | ||
+ | <p>2a)We added 1ul supernatant includes LALF into left plate and 1ul supernatant without LALF into right plate.</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/9/9a/4saat_b.jpg"></p> | ||
+ | <p>2b)We added 100ul supernatant includes LALF into left plate and 100 ul supernatant without LALF into right plate.</p> | ||
+ | <p> </p> | ||
+ | <p> </p> | ||
+ | <p> </p> | ||
+ | <p>Plates were spreaded after 8 hours later;</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/e/e7/8_saat_a.jpg"></p> | ||
+ | <p>3a)We added 1ul supernatant includes LALF into left plate and 1ul suoernatant without LALF into right plate.</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/c/ca/8_saat_b.jpg"></p> | ||
+ | <p>3b)We added 10 ul supernatant includes LALF into left plate and 10 ul supernatant without LALF into right plate.</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/d/de/8_saat_c.jpg"></p> | ||
+ | <p>3c) We added 100ul supernatant includes LALF into left plate and 100 ul supernatant without LALF into right plate.</p> | ||
+ | <p> </p> | ||
+ | <p> </p> | ||
+ | <p>Plates were spreaded after 12 hours later;</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/1/19/12_saat_a.jpg"></p> | ||
+ | <p>4a) We added 1ul supernatant includes LALF into left plate and 1ul suoernatant without LALF into right plate.</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/2/2b/12_saat_b.jpg"></p> | ||
+ | <p>4b)We added 10 ul supernatant includes LALF into left plate and 10 ul supernatant without LALF into right plate.</p> | ||
+ | <p> </p> | ||
+ | <p><img src="https://static.igem.org/mediawiki/igem.org/f/fc/12_saat_c.jpg"></p> | ||
+ | <p>4c)We added 100ul supernatant includes LALF into left plate and 100 ul supernatant without LALF into right plate.</p> | ||
+ | <p><em><br /> | ||
+ | </em></p> | ||
- | <h3><strong>Experiment of liquid culture</strong></h3><br> | + | <h3><strong><span id="8">Experiment of liquid culture</span></strong></h3><br> |
We wanted to see the effects of supernatant of B.subtilis which produces LALF in order to stop e.coli with RFP growth in liquid culture.<br> | We wanted to see the effects of supernatant of B.subtilis which produces LALF in order to stop e.coli with RFP growth in liquid culture.<br> | ||
We put 8 ml LB broth in falcons.<br> | We put 8 ml LB broth in falcons.<br> | ||
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<hr><br> | <hr><br> | ||
- | <p><strong><span style="text-decoration: underline;">CHARACTERIZATION OF LALF PROTEIN</span></strong></p> | + | <p><strong><span style="text-decoration: underline;" id="C">CHARACTERIZATION OF LALF PROTEIN</span></strong></p> |
<p> </p> | <p> </p> | ||
<p><span style="text-decoration: underline;">TARGET</span>: Showing that the LALF protein is produced.</p> | <p><span style="text-decoration: underline;">TARGET</span>: Showing that the LALF protein is produced.</p> | ||
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</div> | </div> |
Revision as of 01:26, 22 September 2011
2011 © Fatih Medical School