Difference between revisions of "Team:DTU-Denmark/Project/Tyrocidine"

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         Introduction to Tyrocidine
 
         Introduction to Tyrocidine
 
       </h1>
 
       </h1>
−
       <p>Tyrocidine is a mixture of non-ribosomal antibiotics, naturally expressed by <i>Brevibacillus parabrevis. </i>By expressing in <i>Bacillus subtilis, </i>tyrocidine becomes accessible for improvement by oligo mediated recombineering and analogues can be screened for reduced toxicity.</p>
+
       <p>Tyrocidine is a mixture of non-ribosomal antibiotics, naturally expressed by <i>Brevibacillus parabrevis. </i>Due to its toxicity, tyrocidine is only suited for topical use. We focused on&nbsp;expressing the&nbsp;non-ribosomal peptide synthetase in&nbsp;<i>Bacillus subtilis, </i>an established model organism, and thereby making it&nbsp;accessible for improvement by oligo mediated recombineering. By using recombineering to alter the active site determining the substrate specificity, new variants of the antibiotic can be created which could be screened for reduced toxicity.</p>
  
 
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         Achievements
 
         Achievements
 
       </h1>
 
       </h1>
−
       <p>We have succeeded assembling all parts except for the repressor. Read more under [MISSING LINK]. When the promoter can be expressed, the construct becomes a promising tool for the expression of the antibiotic tyrocidine.&nbsp;</p>
+
       <ul>
−
 
+
<li>All parts except for the repressor have been assembled. Read more under [MISSING LINK]. When the promoter can be expressed, the construct becomes a promising tool for the expression of the antibiotic tyrocidine.&nbsp;</li>
−
<p>We have established a protocol to&nbsp;<a href="http://dtuwiki-drewt.rhcloud.com/Team:DTU-Denmark/Project/Detection">detect tyrocidine</a> which has been tested by using commercially purchased tyrocidine.</p>
+
<li>A protocol to&nbsp;<a href="http://dtuwiki-drewt.rhcloud.com/Team:DTU-Denmark/Project/Detection">detect tyrocidine</a>&nbsp;has been established&nbsp;which has been tested by using commercially purchased tyrocidine.</li>
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</ul>
  
 
     </div>
 
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         Background
 
         Background
 
       </h1>
 
       </h1>
−
       <p>Tyrocidine is a mixture of non-ribosomal, cyclic antibiotics produced by <img alt="" src="None" style="float: right; width: 401px; height: 325px;" /><br />
+
       <table align="right" border="0" cellpadding="0" cellspacing="0">
−
<i>Brevibacillus parabrevis</i> and has a unique mode of action wherein it disrupts the function of the cell membrane. Tyrocidine consists of four decapeptides varying at three amino acids (MISSING Table #) which are produced by an NRPS system which is capable of incorporating structurally similar amino acids at position 3, 4 and 7 [1]. The three enzymes Tyrocidine Synthetase A, B and C contain 1, 3, and 6 modules, respectively, and assemble Tyrocidine in an assembly line manner (Figure # MISSING HIGHER UP MICHAEL).</p>
+
<thead>
 +
<tr>
 +
<th colspan="4">
 +
<table border="0" cellpadding="0" cellspacing="0">
 +
<tbody>
 +
<tr>
 +
<td><strong>Table 1&nbsp;</strong>&nbsp;Tyrocidine variability.</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</th>
 +
</tr>
 +
<tr>
 +
<th>&nbsp;</th>
 +
<th colspan="3">
 +
<p><strong>Amino acid position</strong></p>
 +
</th>
 +
</tr>
 +
<tr>
 +
<th>
 +
<p><strong>Tyrocidine</strong></p>
 +
</th>
 +
<th>
 +
<p><strong>3</strong></p>
 +
</th>
 +
<th>
 +
<p><strong>4</strong></p>
 +
</th>
 +
<th>
 +
<p><strong>7</strong></p>
 +
</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td>
 +
<p><strong>A</strong></p>
 +
</td>
 +
<td>
 +
<p>L-Phe</p>
 +
</td>
 +
<td>
 +
<p>D-Phe</p>
 +
</td>
 +
<td>
 +
<p>L-Tyr</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><strong>B</strong></p>
 +
</td>
 +
<td>
 +
<p>L-Trp</p>
 +
</td>
 +
<td>
 +
<p>D-Phe</p>
 +
</td>
 +
<td>
 +
<p>L-Tyr</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><strong>C</strong></p>
 +
</td>
 +
<td>
 +
<p>L-Trp</p>
 +
</td>
 +
<td>
 +
<p>D-Trp</p>
 +
</td>
 +
<td>
 +
<p>L-Tyr</p>
 +
</td>
 +
</tr>
 +
<tr>
 +
<td>
 +
<p><strong>D</strong></p>
 +
</td>
 +
<td>
 +
<p>L-Trp</p>
 +
</td>
 +
<td>
 +
<p>D-Trp</p>
 +
</td>
 +
<td>
 +
<p>L-Trp</p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p>Tyrocidine is a mixture of non-ribosomal, cyclic antibiotics produced by<br />
 +
<i>Brevibacillus parabrevis</i> and has a unique mode of action wherein it disrupts the function of the cell membrane. Tyrocidine consists of four decapeptides varying at three amino acids&nbsp;which are produced by an NRPS system which is capable of incorporating structurally similar amino acids at position 3, 4 and 7 [1]. The three enzymes Tyrocidine Synthetase A, B and C contain 1, 3, and 6 modules, respectively, and assemble Tyrocidine in an assembly line manner ().</p>
 +
 
 +
<p>Unfortunately, it has high toxicity towards human blood and reproductive cells and can therefore only be used in topical applications&nbsp;[2].&nbsp;This makes tyrocidine an interesting target for drug improvement. Oligo mediated recombineering is a promising tool to create new analogues with reduced toxicity. Under MISSING section you can read more about improvement of tyrocidine.</p>
 +
 
 +
<p>The genes tycA, tycB and tycC encoding the tyrocidine synthetases are located on the tyrocidine operon, which contains three additional open reading frames, labelled tycD, tycE and TycF located downstream of the synthetase genes. The entire operon has a size of 39.5 kb. [3]</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p><img alt="" src="None" style="float: left; width: 401px; height: 325px;" /></p>
 +
 
 +
<p><br />
 +
&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p>&nbsp;</p>
  
−
<p>Unfortunately, it has high toxicity towards human blood and reproductive cells and can therefore only be used in topical applications. This makes tyrocidine an interesting target for drug improvement. Oligo mediated recombineering is a promising tool to create new analogues with reduced toxicity. Under MISSING section you can read more about improvement of tyrocidine.</p>
+
<p><br />
 +
Figure 1: Structure of tyrocidine A</p>
  
−
<p>The genes tycA, tycB and tycC encoding the tyrocidine synthetases are located on the tyrocidine operon, which contains three additional open reading frames, labelled tycD, tycE and TycF located downstream of the synthetase genes. The entire operon has a size of 39.5 kb. [2]</p>
+
<p>&nbsp;</p>
  
 
     </div>
 
     </div>
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       </h1>
 
       </h1>
 
       <p>Due to the big size of the biosynthetic cluster, an amplification with standard PCR is not feasible. We designed a construct for the expression of tyrocidine which can integrate into the lacA site of <i>Bacillus subtilis</i>, an established model organism. By counter-selection with a toxic gene cassette, the tyrocidine operon can insert into the construct by homologous recombination. The toxic gene is placed under control of the Pxyl promoter, which is repressed by a repressor in the absence of xylose. If xylose is present, the repressor leaves the operon and the toxic gene is transcribed.</p>
 
       <p>Due to the big size of the biosynthetic cluster, an amplification with standard PCR is not feasible. We designed a construct for the expression of tyrocidine which can integrate into the lacA site of <i>Bacillus subtilis</i>, an established model organism. By counter-selection with a toxic gene cassette, the tyrocidine operon can insert into the construct by homologous recombination. The toxic gene is placed under control of the Pxyl promoter, which is repressed by a repressor in the absence of xylose. If xylose is present, the repressor leaves the operon and the toxic gene is transcribed.</p>
 +
 +
<div aria-multiselectable="true" class="panel-group" id="accordion4" role="tablist">
 +
<div class="panel panel-default">
 +
<div class="panel-heading" id="headingFour" role="tab">
 +
<h4 class="panel-title"><a aria-controls="collapseFour" aria-expanded="false" class="collapsed" data-parent="#accordion4" data-toggle="collapse" href="#collapseFour" role="button">Cloning of Tyrocidine construct</a></h4>
 +
</div>
 +
 +
<div aria-labelledby="headingFour" class="panel-collapse collapse" id="collapseFour" role="tabpanel">
 +
<div class="panel-body">The parts of the tyrocidine construct were amplified by PCR reaction and the linearized biobrick plasmid was cut by the adequate restriction enzymes. The parts were run on an agarose gel to verify their length and purified before they were assembled by Gibson assembly. The constructs were transformed into chemocompetent E.coli and grown overnight on an LB agar plate with the specific antibiotics. The reaction was controlled by amplifying the tyrocidine construct in the Gibson assembly mix by PCR or by doing a colony PCR of the transformants and running the amplified fragment on a gel.&nbsp;</div>
 +
</div>
 +
</div>
 +
</div>
  
 
     </div>
 
     </div>
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       </h1>
 
       </h1>
 
       <p>Our efforts to express tyrocidine have been hampered by difficulties in assembling all parts. All parts could be assembled except for the xylose repressor, which seemed to have caused troubles in all our experimental designs.</p>
 
       <p>Our efforts to express tyrocidine have been hampered by difficulties in assembling all parts. All parts could be assembled except for the xylose repressor, which seemed to have caused troubles in all our experimental designs.</p>
−
 
−
<h4><a name="h.pvv6evarbfzp"></a><b>Methods for creating a construct for Expression of Tyrocidine</b></h4>
 
−
 
−
<p>The parts of the tyrocidine construct were amplified by PCR reaction and the linearized biobrick plasmid was cut by the adequate restriction enzymes. The parts were run on an agarose gel to verify their length and purified before they were assembled by Gibson assembly. The constructs were transformed into chemocompetent E.coli and grown overnight on an LB agar plate with the specific antibiotics. The reaction was controlled by amplifying the tyrocidine construct in the Gibson assembly mix by PCR or by doing a colony PCR of the transformants and running the amplified fragment on a gel.</p>
 
  
 
     </div>
 
     </div>
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           <li>Lipmann, F., Roskoski, R., Gevers, W., & Kleinkauf, H. (1970). Tyrocidine biosynthesis by three complementary fractions from Bacillus brevis (ATCC 8185). Biochemistry, 9(25), 4839–4845. <a href="http://dx.doi.org/10.1021/bi00827a002" target="_blank">doi:10.1021/bi00827a002</a></li>
 
           <li>Lipmann, F., Roskoski, R., Gevers, W., & Kleinkauf, H. (1970). Tyrocidine biosynthesis by three complementary fractions from Bacillus brevis (ATCC 8185). Biochemistry, 9(25), 4839–4845. <a href="http://dx.doi.org/10.1021/bi00827a002" target="_blank">doi:10.1021/bi00827a002</a></li>
 +
       
 +
          <li>Joo, S.-H. (2012). Cyclic Peptides as Therapeutic Agents and Biochemical Tools. Biomolecules and Therapeutics, 20(1), 19–26. <a href="http://dx.doi.org/10.4062/biomolther.2012.20.1.019" target="_blank">doi:10.4062/biomolther.2012.20.1.019</a></li>
 
          
 
          
 
           <li>Mootz, H.D., & Marahiel, M.A. (1997). The tyrocidine biosynthesis operon of Bacillus brevis: Complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. Journal of Bacteriology, 179(21):6843-50</li>
 
           <li>Mootz, H.D., & Marahiel, M.A. (1997). The tyrocidine biosynthesis operon of Bacillus brevis: Complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. Journal of Bacteriology, 179(21):6843-50</li>

Revision as of 22:38, 18 September 2015


Tyrocidine

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Introduction to Tyrocidine

Tyrocidine is a mixture of non-ribosomal antibiotics, naturally expressed by Brevibacillus parabrevis. Due to its toxicity, tyrocidine is only suited for topical use. We focused on expressing the non-ribosomal peptide synthetase in Bacillus subtilis, an established model organism, and thereby making it accessible for improvement by oligo mediated recombineering. By using recombineering to alter the active site determining the substrate specificity, new variants of the antibiotic can be created which could be screened for reduced toxicity.

Achievements

  • All parts except for the repressor have been assembled. Read more under [MISSING LINK]. When the promoter can be expressed, the construct becomes a promising tool for the expression of the antibiotic tyrocidine. 
  • A protocol to detect tyrocidine has been established which has been tested by using commercially purchased tyrocidine.

Background

Table 1  Tyrocidine variability.
 

Amino acid position

Tyrocidine

3

4

7

A

L-Phe

D-Phe

L-Tyr

B

L-Trp

D-Phe

L-Tyr

C

L-Trp

D-Trp

L-Tyr

D

L-Trp

D-Trp

L-Trp

Tyrocidine is a mixture of non-ribosomal, cyclic antibiotics produced by
Brevibacillus parabrevis and has a unique mode of action wherein it disrupts the function of the cell membrane. Tyrocidine consists of four decapeptides varying at three amino acids which are produced by an NRPS system which is capable of incorporating structurally similar amino acids at position 3, 4 and 7 [1]. The three enzymes Tyrocidine Synthetase A, B and C contain 1, 3, and 6 modules, respectively, and assemble Tyrocidine in an assembly line manner ().

Unfortunately, it has high toxicity towards human blood and reproductive cells and can therefore only be used in topical applications [2]. This makes tyrocidine an interesting target for drug improvement. Oligo mediated recombineering is a promising tool to create new analogues with reduced toxicity. Under MISSING section you can read more about improvement of tyrocidine.

The genes tycA, tycB and tycC encoding the tyrocidine synthetases are located on the tyrocidine operon, which contains three additional open reading frames, labelled tycD, tycE and TycF located downstream of the synthetase genes. The entire operon has a size of 39.5 kb. [3]

 


 

 

 

 

 

 

 

 

 


Figure 1: Structure of tyrocidine A

 

Experimental Design

Due to the big size of the biosynthetic cluster, an amplification with standard PCR is not feasible. We designed a construct for the expression of tyrocidine which can integrate into the lacA site of Bacillus subtilis, an established model organism. By counter-selection with a toxic gene cassette, the tyrocidine operon can insert into the construct by homologous recombination. The toxic gene is placed under control of the Pxyl promoter, which is repressed by a repressor in the absence of xylose. If xylose is present, the repressor leaves the operon and the toxic gene is transcribed.

The parts of the tyrocidine construct were amplified by PCR reaction and the linearized biobrick plasmid was cut by the adequate restriction enzymes. The parts were run on an agarose gel to verify their length and purified before they were assembled by Gibson assembly. The constructs were transformed into chemocompetent E.coli and grown overnight on an LB agar plate with the specific antibiotics. The reaction was controlled by amplifying the tyrocidine construct in the Gibson assembly mix by PCR or by doing a colony PCR of the transformants and running the amplified fragment on a gel. 

Discussion

Our efforts to express tyrocidine have been hampered by difficulties in assembling all parts. All parts could be assembled except for the xylose repressor, which seemed to have caused troubles in all our experimental designs.

References

  1. Lipmann, F., Roskoski, R., Gevers, W., & Kleinkauf, H. (1970). Tyrocidine biosynthesis by three complementary fractions from Bacillus brevis (ATCC 8185). Biochemistry, 9(25), 4839–4845. doi:10.1021/bi00827a002
  2. Joo, S.-H. (2012). Cyclic Peptides as Therapeutic Agents and Biochemical Tools. Biomolecules and Therapeutics, 20(1), 19–26. doi:10.4062/biomolther.2012.20.1.019
  3. Mootz, H.D., & Marahiel, M.A. (1997). The tyrocidine biosynthesis operon of Bacillus brevis: Complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. Journal of Bacteriology, 179(21):6843-50
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