Difference between revisions of "Part:BBa K2278022"

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__NOTOC__
 
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<partinfo>BBa_K2278022 short</partinfo>
 
<partinfo>BBa_K2278022 short</partinfo>
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=='''Introduction'''==
 
=='''Introduction'''==
 
<html>
 
<html>
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This DNA biobrick was designed in order to produce Lecrocin I AMP in a yeast organism <i></i> strain.  
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This DNA biobrick was designed in order to produce Leucrocin I antimicrobial peptide.  
  
 
<h3 id="RT"> 1- Biological background </h3>
 
<h3 id="RT"> 1- Biological background </h3>
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+
Antimicrobial peptides (AMP) are phylogenetically ancient components of the innate defense of both invertebrates and vertebrates. In the context of growing bacterial antibiotic-resistance, these AMP are considered as potential new therapeutical candidates.  
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+
<br>
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Mechanisme
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Leucrocin I from Siamese crocodile white blood cells shows a good antibacterial activity towards <i>Vibrio cholerae</i>.
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Antimicrobial peptides are phylogenitically ancient components of innate defense mechanisms of both invertebrates and vertebrates. In the context of growing prevalence of antibiotic-resistance of bacterial strain, the AMP can be considered as potential new therapeutical candidates.  
+
Leucrocin I action has been observed with fluorescence and electron microscopy. The molecule is cationic and can target bacterial membranes. It creates pores in these membranes leading to the cell lysis.
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+
−
 
+
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Leucrocin I from Siamese crocodile white blood cells shows a good antibacterial activity towards Vibrio cholerae.  
+
 
The peptide is a 7 amino acid residue : NGVQPKY with a molecular mass around 806.99 Da.  
 
The peptide is a 7 amino acid residue : NGVQPKY with a molecular mass around 806.99 Da.  
−
 
−
The mechanism of action of the Leucrocin I has been observed with fluorescence and electron microscopy This cationic molecules and can target bacterium membranes, to create pores in it, leading to the lysis of the cells.
 
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<figure><p style="text-align:center;"> <img src ="https://static.igem.org/mediawiki/parts/4/4d/Yaraksa14.png" width = "600" /> <figcaption> Figure 1: <b>Scanning electron micrographs of Vibrio cholerae treated with peptides </b>  (a) control control bacteria c) bacteria treated with AMPs (Yaraksa and al., 2014)</figcaption> </figure>
 
  
 
<h3 id="RT"> 2- Usage in iGEM projects </h3>
 
<h3 id="RT"> 2- Usage in iGEM projects </h3>
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+
The part was designed during the Croc’n Cholera project <a href="http://2017.igem.org/Team:INSA-UPS_France">(team INSA-UPS-France 2017)</a>. It produces the leucrocin I AMP when associated with a yeast promoter. The α-factor (</html><partinfo>BBa_K1800001</partinfo>)<html> sequence contains a RBS and a signal sequence to secrete the produced peptides.
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The part was designed to  constitutively produce the leucrocin I AMP with a yeast promoter. The α-factor (BBa_K1800001) sequence contains a RBS and a signal sequence to secrete the produced peptides.  
+
−
 
+
 
</html>
 
</html>
 +
<br>
 +
<br>
  
 
=='''Experiments'''==
 
=='''Experiments'''==
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<h3 id="RT"> 1- Molecular biology </h3>
 
<h3 id="RT"> 1- Molecular biology </h3>
 
<p>
 
<p>
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The gene was placed in silico under the control of an alpha factor signal. IDT performed the DNA synthesis and delivered the part as gBlock. 
+
The gene was placed under the control of an alpha factor signal. IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into the pSB1C3 plasmid.
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+
−
The construct was cloned by conventional ligation into pSB1C3 plasmid  
+
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The construction was then inserted on plasmid pPICZa and integrated in the yeast genome.
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</p>
 
</p>
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<br>
 
<b>Analysis of the restriction map </b>
 
<b>Analysis of the restriction map </b>
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/1/1e/LeucroI-gel.png" width = "400"/><figcaption> Figure 2: <b>Analysis of the restriction map BBa_K2278022 in pSB1C3.</b> Digested plasmids are electrophoresed through an 0.7% agarose gel. The desired plasmids lengths are pSB1C3 (2029bp) the other band correspond to a 300bp insert.</figcaption></figure>
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</html>
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[[Image:T--INSA-UPS_France--LeucroI-gel2.png|800px|thumb|center|'''Figure 1:''' <b>Analysis of the restriction map BBa_K2278022. </b> Digested fragments (Xba1 and Pst1) are electrophoresed through a 1% agarose gel. Control vector pSB1C3 contained an insert and expected size were 2034 and 700 bp (digestion was not total, hence the 2734 bp fragment). The fragment lengths of the tested clone were 2035 bp and 319 bp for the required insert.]]
  
 +
<html>
 +
 +
<br>
 
<p><b>Sequencing </p></b>
 
<p><b>Sequencing </p></b>
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/9/93/Leucroseq.png" width = "700"/><figcaption> Figure 3: <b>Sequencing  of pSB1C3+ BBa_K2278022 </b> 1500 ng of plasmid are sequenced. 1 oligo was used to perform the sequencing. The obtained sequence were blast on the BBa_K2278022 sequence with the iGEM sequencing online tools. </figcaption></figure>
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</html>
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[[Image:T--INSA-UPS_France--leucroseq.png|800px|thumb|center|'''Figure 2:''' <b>Sequencing  of pSB1C3-Leucrocin I.</b> 1500 ng of plasmid are sequenced. The obtained sequence were blast on the BBa_K2278022 sequence with the iGEM sequencing online tools. ]]
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 +
<html>
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The sequencing successfully validated the sequence of the biobrick.  
 
The sequencing successfully validated the sequence of the biobrick.  
 +
<br>
  
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<h3 id="RT"> 2- Expression  <i>in vivo</i>  </h3>
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<h3 id="RT"> 2- Integration in <i>Pichia pastoris</i>  </h3>
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<p><b>Integration in Pichia pastori genome </b></p>
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The biobrick was placed under the control of the methanol inducible pAOXI promoter (</html><partinfo>BBa_K431007</partinfo><html>) and was cloned in the pPICZalpha vector, an expression vector for the yeast Pichia pastoris.
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<p> Protocole </p>
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The plasmid was then linearized and transferred in <i>Pichia pastoris</i> by electroporation. The integration is done by using pAOXI homology region (present on pPICZalpha). Indeed, the pAOXI promoter made genome recombination easier in <i>Pichia pastoris</i>.  
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The biobrick was placed in silico under the control of p(GAP) promoter (BBa_K431009) and was cloned in pPICZalpha vector, a good expression vector for Pichia pastoris.  
+
  
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The plasmid was then linearized and transferred in Pichia pastoris by electroporation. The integration is predicted to be at the p(GAP) location. Indeed, the p(GAP) promoter makes genome recombination easier in Pichia pastoris genome thanks to its homology site.
 
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/4/41/IntegrationAMP.png"/><figcaption> Figure 3: <b>Integration of p(GAP)+BBa_K2278022 in pichia pastoris </b> To verify the function of the new Biobrick, we performed a DNA extraction. To check the length of the resulting DNA, we digested the DNA with EcoRI and Ncos restriction enzyme and electrophoresed the reactions through an 0,7% agarose gel. Lane 1 correspond to 1kb DNA ladder (new England bolas, Inc)
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</html>
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</figcaption></figure>
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[[Image:T--INSA-UPS_France--11-08_leucro_pcr_colonies_-_copie_2.png|800px|thumb|center|'''Figure 3:''' <b>Integration of pGAP+BBa_K2278022 in <i>Pichia pastoris</i></b>. To verify the correct integration, we performed colony PCR and electrophoresed the product through a 1% agarose gel. A 241 bp fragment is expected if the integration is correct. ]]
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 +
<html>
 +
 
 +
Correct amplifications were observed for the 2 tested colonies A & B (in spite of lower size non-specific fragments). Negative control with pPICZalpha did not present the correct size fragment, as expected.
 +
<br><br>
 +
 
  
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<p><b>Expression of Leucrocin I AMP </b></p>
 
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Leucrocin I  production was performed with the P. pastoris YPD 40 g/L glucose and grown for 4 days at 30 °C in an agitating incubator. 15mL of each supernatant culture were stored at 4°C while 35mL were freeze-dried and then resuspended in 3.5mL of water.
 
  
 
</html>
 
</html>
  
 
=='''Characterization'''==
 
=='''Characterization'''==
 +
<html>
 +
<h3 id="RT">2. Toxicity assay  </h3>
 +
 +
Leucrocin  production was performed with Pichia pastoris rown for 4 days at 30 °C with shacking in YPD 40 g/L glucose plus methanol to trigger the pAOX1 promoter. Supernatants from yeasts with or without the leucrocin encoding gene were sampled. The supernatants were used in a halo assay against <i>V. harveyi</i> as the target of Leucrocin. Briefly, 35mL of supernatants were freeze-dried and then resuspended in 3.5mL of water. A paper cut was soaked with one of these solutions and placed on a Petri plate inoculated with <i>V. harveyi</i> (figure 3).
 +
 +
</html>
 +
[[Image:T--INSA-UPS_France--LeucrocinI.png|800px|thumb|center|'''Figure 4:''' <b>AMP halo assay.</b> Positive control was performed with chloramphenicol (25 g/L), the negative control was performed with the empty plasmid integrated in <i>P. pastoris</i>, the assay was performed with the plasmid containing BBa_K2278022 integrated in <i>P. pastoris</i>.]]
  
 
<html>
 
<html>
  
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<h3 id="RT"> Toxicity assay </h3>
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<p><b>Conclusion :  </b> </p>
 +
No inhibition halo was observed around the yeast patch. The Leucrocin I cytoxicity can not be demonstrated.
 +
<p><b>Perspectives: </b></p>
 +
Higher concentration of yeast supernatants could be tried.
 +
<br>
 +
<br>
 +
</html>
 +
===Design Notes===
 +
 
 +
<partinfo>BBa_K1800001</partinfo> : Alpha-Factor Secretion Signal
 +
 
 +
===Source===
 +
 
 +
The peptides DNA sequence has been obtained by reverse translate the amino acid sequence of the Leucrocin I proposed by Pata <i>et al</i>., 2011.  Pata <i>et al</i>., 2011 had determinated the amino acid sequence  by mass spectrometry analysis.
  
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The engineered yeast were used in a halo assay against V. harveyi as the target of AMPs. A paper soacked with a yeast solution was placed on the plate and V. harveyi growth in the viscinity of the yeast patch was followed.
 
  
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The toxicity assay did not reveal any activity of the Leucrocin I AMP
+
===References===
 +
Pata, S., Yaraksa, N., Daduang, S., Temsiripong, Y., Svasti, J., Araki, T. and Thammasirirak, S. (2011). Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Developmental & Comparative Immunology, 35(5), pp.545-553.

Latest revision as of 10:56, 30 October 2017

Lecrocin I antimicrobial peptide with Alpha-Factor Secretion Signal

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 244
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

This DNA biobrick was designed in order to produce Leucrocin I antimicrobial peptide.

1- Biological background

Antimicrobial peptides (AMP) are phylogenetically ancient components of the innate defense of both invertebrates and vertebrates. In the context of growing bacterial antibiotic-resistance, these AMP are considered as potential new therapeutical candidates.
Leucrocin I from Siamese crocodile white blood cells shows a good antibacterial activity towards Vibrio cholerae. Leucrocin I action has been observed with fluorescence and electron microscopy. The molecule is cationic and can target bacterial membranes. It creates pores in these membranes leading to the cell lysis. The peptide is a 7 amino acid residue : NGVQPKY with a molecular mass around 806.99 Da.

2- Usage in iGEM projects

The part was designed during the Croc’n Cholera project (team INSA-UPS-France 2017). It produces the leucrocin I AMP when associated with a yeast promoter. The α-factor (BBa_K1800001) sequence contains a RBS and a signal sequence to secrete the produced peptides.

Experiments

1- Molecular biology

The gene was placed under the control of an alpha factor signal. IDT performed the DNA synthesis and delivered the part as gBlock. The construct was cloned by conventional ligation into the pSB1C3 plasmid.


Analysis of the restriction map

Figure 1: Analysis of the restriction map BBa_K2278022. Digested fragments (Xba1 and Pst1) are electrophoresed through a 1% agarose gel. Control vector pSB1C3 contained an insert and expected size were 2034 and 700 bp (digestion was not total, hence the 2734 bp fragment). The fragment lengths of the tested clone were 2035 bp and 319 bp for the required insert.


Sequencing

Figure 2: Sequencing of pSB1C3-Leucrocin I. 1500 ng of plasmid are sequenced. The obtained sequence were blast on the BBa_K2278022 sequence with the iGEM sequencing online tools.

The sequencing successfully validated the sequence of the biobrick.

2- Integration in Pichia pastoris

The biobrick was placed under the control of the methanol inducible pAOXI promoter (BBa_K431007) and was cloned in the pPICZalpha vector, an expression vector for the yeast Pichia pastoris. The plasmid was then linearized and transferred in Pichia pastoris by electroporation. The integration is done by using pAOXI homology region (present on pPICZalpha). Indeed, the pAOXI promoter made genome recombination easier in Pichia pastoris.

Figure 3: Integration of pGAP+BBa_K2278022 in Pichia pastoris. To verify the correct integration, we performed colony PCR and electrophoresed the product through a 1% agarose gel. A 241 bp fragment is expected if the integration is correct.

Correct amplifications were observed for the 2 tested colonies A & B (in spite of lower size non-specific fragments). Negative control with pPICZalpha did not present the correct size fragment, as expected.

Characterization

2. Toxicity assay

Leucrocin production was performed with Pichia pastoris rown for 4 days at 30 °C with shacking in YPD 40 g/L glucose plus methanol to trigger the pAOX1 promoter. Supernatants from yeasts with or without the leucrocin encoding gene were sampled. The supernatants were used in a halo assay against V. harveyi as the target of Leucrocin. Briefly, 35mL of supernatants were freeze-dried and then resuspended in 3.5mL of water. A paper cut was soaked with one of these solutions and placed on a Petri plate inoculated with V. harveyi (figure 3).

Figure 4: AMP halo assay. Positive control was performed with chloramphenicol (25 g/L), the negative control was performed with the empty plasmid integrated in P. pastoris, the assay was performed with the plasmid containing BBa_K2278022 integrated in P. pastoris.

Conclusion :

No inhibition halo was observed around the yeast patch. The Leucrocin I cytoxicity can not be demonstrated.

Perspectives:

Higher concentration of yeast supernatants could be tried.

Design Notes

BBa_K1800001 : Alpha-Factor Secretion Signal

Source

The peptides DNA sequence has been obtained by reverse translate the amino acid sequence of the Leucrocin I proposed by Pata et al., 2011. Pata et al., 2011 had determinated the amino acid sequence by mass spectrometry analysis.


References

Pata, S., Yaraksa, N., Daduang, S., Temsiripong, Y., Svasti, J., Araki, T. and Thammasirirak, S. (2011). Characterization of the novel antibacterial peptide Leucrocin from crocodile (Crocodylus siamensis) white blood cell extracts. Developmental & Comparative Immunology, 35(5), pp.545-553.