Difference between revisions of "Team:Montpellier/Model"

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        <title>Modeling</title>
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<h1>Modeling</h1><hr/>
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<p>Bacteria such as <i>E. coli</i> are very well characterized, and we know the features and the typical nucleotide patterns that has to be modified in order to tune gene expression. However, <i>L. jensenii</i> is a little-known bacterium and we only recently started to engineer its genome for biomedical applications [1]. With this study we propose a general pipeline to identify promoters found in the genome of <i>L. jensenii</i>, a first step towards the characterisation of this organism.</p>
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<p>The aim of our modeling was to predict which natural pre-gene sequences from <i>L. jensenii</i> were most likely to be to be strong promoters. For this we used bioinformatic tools to identify recognizable patterns in those pre-gene sequences such as the Shine Dalgarno sequence or minus 10 and minus 35 sequences from promoters typical of this bacteria. Then we selected the pre-gene sequences having those patterns to test their promoter force with a signal of RFP. We wanted to find natural sequences with different promoter forces, in the goal to build a toolbox of promoters sequences for <i>L. jensenii</i>.</p>
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<p>First, we needed to extract those pre-gene sequences from the full genome. We downloaded it from NCBI (<i>L. jensenii</i> JV-V16). For this we followed the protocol of the following article that had the goal to identify promoter sequences on <i>L. plantarum</i> : "Genome-wide prediction and validation of sigma70 promoters in <i>Lactobacillus plantarum WCFS1</i>”. We developed a python script to do it. Selecting a maximum length of 100bp, a minimum of 25bp and no overlap, upstream each gene sequence.<p>
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<p>Then we used the “MEME SUITE: tools for motif discovery and searching” to identify relevant patterns on those sequences. With the results we were able to identify different known patterns present on multiples pre-gene sequences (number of hits):</p>
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 +
<p>The RBS pattern of the bacteria, a Shine Dalgarno sequence specific of <i>L. jensenii</i>:
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<figure>
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<img class="" src=""/><br/>
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<figcaption><span class="underline">Figure 1:</span> JV-V16 Shine Dalgarno (RBS) motif with 531 hits on MEME.</figcaption>
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</figure>
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</p>
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<p>The pattern of <i>L. jensenii</i> promoters:
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<figure>
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<img class="" src=""/><br/>
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<figcaption><span class="underline">Figure 2:</span> JV-V16 Promoter motif for 176 hits.</figcaption>
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</figure>
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</p>
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 +
<p>We can clearly see the -10 pattern: TATAAT which resembles to some of other <i>Lactobacilli</i> patterns.</p>
 +
 +
<p>Furthermore we identified the -35 pattern by selecting the sequences having a promoter pattern for an other MEME run:
 +
<figure>
 +
<img class="" src=""/><br/>
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<figcaption><span class="underline">Figure 3:</span> JV-V16 -35 motif with 42 hits.</figcaption>
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</figure>
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</p>
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<p>We also found pattern looking like terminators tails:
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<figure>
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<img class="" src=""/><br/>
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<figcaption><span class="underline">Figure 4:</span> Motif of the tails of promoters for 222 hits.</figcaption>
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</figure>
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</p>
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<p>With those informations on what RBS, promoters and terminator tails looked like on <i>L. jensenii</i> we then ran a MAST (MEME SUITE) to found those patterns on our pre-genes sequences. We then selected some to test their promoters forces with a RFP signal, we took 4 with RBS and promoter patterns, 3 with only promoter patterns, 2 with only RBS pattern, and finally one without any. Plus, we created an artificial sequence with promoter and RBS with the most common letter from the power weight matrice of each patterns. We also took a characterized promoter from jensenii as positive control, and a sequence with only RFP as negative control.</p>
 +
 +
<p>We then cloned those sequences on the plasmid for <i>L. jensenii</i> (pLEM415) and prepared them to be transformed on L.jensenii. Sadly their were some issue with the transformation with <i>L. jensenii</i> and we were not able to have results on this part of the project.</p>
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</section>
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<section class="references" id="references">
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  <table class="references_table">
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    <tr>
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      <th class="references_title" colspan="2">References</th>
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    </tr>
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    <tr>
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      <td class="references_left">[1]</td>
 +
      <td class="references_right">Rajesh K. Naz, Subhash C.Chauhan. 2001. Presence of antibodies to sperm YLP12 synthetic peptide in sera and seminal plasma of immunoinfertile men. <i>Molecular Human Reproduction</i> Vol.7 no.1 pp. 21–26.</td>
 +
    </tr>
 +
    <tr>
 +
      <td class="references_left">[2]</td>
 +
      <td class="references_right">Rajesh K. Naz. 2014. Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide. <i>Protein Science</i> 2014 Vol.23:857—868.</td>
 +
    </tr>
 +
    <tr>
 +
      <td class="references_left">[3]</td>
 +
      <td class="references_right">A.S. Samuel and R.K. Naz. 2008. Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development. <i>Human Reproduction</i> Vol.23, No.6 pp. 1324–1337.</td>
 +
    </tr>
 +
    <tr>
 +
      <td class="references_left">[4]</td>
 +
      <td class="references_right">Angela Marcobal, Xiaowen Liu, Wenlei Zhang, Antony S. Dimitrov, Letong Jia, Peter P. Lee, Timothy R. Fouts, Thomas P. Parks, and Laurel A. Lagenaur. 2016. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal <i>Lactobacillus</i>. <i>Aids Resaerch And Human Retroviruses</i> Volume 32, Number 10/11.</td>
 +
    </tr>
 +
    <tr>
 +
      <td class="references_left">[5]</td>
 +
      <td class="references_right">Xiaowen Liu, Laurel A. Lagenaur, David A. Simpson, Kirsten P. Essenmacher, Courtney L. Frazier-Parker, Yang Liu, Daniel Tsai, Srinivas S. Rao, Dean H. Hamer, Thomas P. Parks, Peter P. Lee and Qiang Xu. 2006. Engineered Vaginal <i>Lactobacillus</i> Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N. <i>Antimicrob Agents Chemother</i> Vol. 50, No. 10, p. 3250–3259</td>
 +
    </tr>
 +
    <tr>
 +
      <td class="references_bottom_left">[6]</td>
 +
      <td class="references_bottom_right">Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., ... & Rout, M. P. 2014. A robust pipeline for rapid production of versatile nanobody repertoires. <i>Nature methods</i>, 11(12), 1253.</td>
 +
    </tr>
 +
  </table>
  
 
</section>
 
</section>

Revision as of 21:01, 10 October 2018

Modeling

Modeling

Bacteria such as E. coli are very well characterized, and we know the features and the typical nucleotide patterns that has to be modified in order to tune gene expression. However, L. jensenii is a little-known bacterium and we only recently started to engineer its genome for biomedical applications [1]. With this study we propose a general pipeline to identify promoters found in the genome of L. jensenii, a first step towards the characterisation of this organism.

The aim of our modeling was to predict which natural pre-gene sequences from L. jensenii were most likely to be to be strong promoters. For this we used bioinformatic tools to identify recognizable patterns in those pre-gene sequences such as the Shine Dalgarno sequence or minus 10 and minus 35 sequences from promoters typical of this bacteria. Then we selected the pre-gene sequences having those patterns to test their promoter force with a signal of RFP. We wanted to find natural sequences with different promoter forces, in the goal to build a toolbox of promoters sequences for L. jensenii.

First, we needed to extract those pre-gene sequences from the full genome. We downloaded it from NCBI (L. jensenii JV-V16). For this we followed the protocol of the following article that had the goal to identify promoter sequences on L. plantarum : "Genome-wide prediction and validation of sigma70 promoters in Lactobacillus plantarum WCFS1”. We developed a python script to do it. Selecting a maximum length of 100bp, a minimum of 25bp and no overlap, upstream each gene sequence.

Then we used the “MEME SUITE: tools for motif discovery and searching” to identify relevant patterns on those sequences. With the results we were able to identify different known patterns present on multiples pre-gene sequences (number of hits):

The RBS pattern of the bacteria, a Shine Dalgarno sequence specific of L. jensenii:


Figure 1: JV-V16 Shine Dalgarno (RBS) motif with 531 hits on MEME.

The pattern of L. jensenii promoters:


Figure 2: JV-V16 Promoter motif for 176 hits.

We can clearly see the -10 pattern: TATAAT which resembles to some of other Lactobacilli patterns.

Furthermore we identified the -35 pattern by selecting the sequences having a promoter pattern for an other MEME run:


Figure 3: JV-V16 -35 motif with 42 hits.

We also found pattern looking like terminators tails:


Figure 4: Motif of the tails of promoters for 222 hits.

With those informations on what RBS, promoters and terminator tails looked like on L. jensenii we then ran a MAST (MEME SUITE) to found those patterns on our pre-genes sequences. We then selected some to test their promoters forces with a RFP signal, we took 4 with RBS and promoter patterns, 3 with only promoter patterns, 2 with only RBS pattern, and finally one without any. Plus, we created an artificial sequence with promoter and RBS with the most common letter from the power weight matrice of each patterns. We also took a characterized promoter from jensenii as positive control, and a sequence with only RFP as negative control.

We then cloned those sequences on the plasmid for L. jensenii (pLEM415) and prepared them to be transformed on L.jensenii. Sadly their were some issue with the transformation with L. jensenii and we were not able to have results on this part of the project.

References
[1] Rajesh K. Naz, Subhash C.Chauhan. 2001. Presence of antibodies to sperm YLP12 synthetic peptide in sera and seminal plasma of immunoinfertile men. Molecular Human Reproduction Vol.7 no.1 pp. 21–26.
[2] Rajesh K. Naz. 2014. Vaccine for human contraception targeting sperm Izumo protein and YLP12 dodecamer peptide. Protein Science 2014 Vol.23:857—868.
[3] A.S. Samuel and R.K. Naz. 2008. Isolation of human single chain variable fragment antibodies against specific sperm antigens for immunocontraceptive development. Human Reproduction Vol.23, No.6 pp. 1324–1337.
[4] Angela Marcobal, Xiaowen Liu, Wenlei Zhang, Antony S. Dimitrov, Letong Jia, Peter P. Lee, Timothy R. Fouts, Thomas P. Parks, and Laurel A. Lagenaur. 2016. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus. Aids Resaerch And Human Retroviruses Volume 32, Number 10/11.
[5] Xiaowen Liu, Laurel A. Lagenaur, David A. Simpson, Kirsten P. Essenmacher, Courtney L. Frazier-Parker, Yang Liu, Daniel Tsai, Srinivas S. Rao, Dean H. Hamer, Thomas P. Parks, Peter P. Lee and Qiang Xu. 2006. Engineered Vaginal Lactobacillus Strain for Mucosal Delivery of the Human Immunodeficiency Virus Inhibitor Cyanovirin-N. Antimicrob Agents Chemother Vol. 50, No. 10, p. 3250–3259
[6] Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., ... & Rout, M. P. 2014. A robust pipeline for rapid production of versatile nanobody repertoires. Nature methods, 11(12), 1253.

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Centre de Biochimie Structurale, 29 Rue de Navacelles, 34090 Montpellier, France