Difference between revisions of "Part:BBa K4645004"

m
m (→‎Characterization)
 
(22 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4645004 short</partinfo>
 
<partinfo>BBa_K4645004 short</partinfo>
 +
<br>
 +
In complex with DPO, VqmA<sub>phage</sub> binds to the qtip promoter (<bbpart>BBa_K4645005</bbpart>), activating transcription.
 +
Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA, which together repress genes for virulence and biofilm formation [1]. VqmA<sub>Vc</sub> activates expression of the <i>vqmR</i> gene encoding the small regulatory RNA VqmR. <i>Vibriophage</i> VP882 encodes a VqmA homolog called VqmA<sub>Phage</sub> that activates the qtip promoter (P<sub>qtip</sub>). It’s worth noting that, VqmA<sub>Phage</sub> can activate <i>vqmR</i> expression but VqmA<sub>Vc</sub> cannot activate expression of qtip. Promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the <i>vqmR</i> and qtip promoters are required to maintain specificity. [2]<br>
  
−
This part is meant to express vqmA gene which would translate into protein VqmA<sub>Phage</sub>.<br>
 
−
Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA, which together repress genes for virulence and biofilm formation. VqmA<sub>Vc</sub> activates expression of the vqmR gene encoding the small regulatory RNA VqmR. Vibriophage VP882 encodes a VqmA homolog called VqmA<sub>Phage</sub> that activates the qtip promoter (Pqtip). It’s worth noting that, VqmA<sub>Phage</sub> can activate vqmR expression but VqmA<sub>Vc</sub> cannot activate expression of qtip. Promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity.
 
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
<p>We assembled this part with P<sub>qtip</sub> then transfected into E. coli to sensing the density of bacteria and regulate the circuit.</p>
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
<title>What？</title>
 +
</head>
 +
<body>
 +
<center><img src=" https://static.igem.wiki/teams/4645/wiki/quorum-sensing/vqma-usage-and.png" style="width:75%; "></center>
 +
<br>
 +
<center><b>Figure 1. The circuit we assembled.</b></center>
 +
<br>
 +
</body>
 +
</html>
 +
 +
===Characterization===
 +
<b>Successful protein expression</b>
 +
<p>We transformed <i>vqmA</i> into <i>E.coli</i> BL21(DE3) and cultivated in 37℃, 220 rpm. When the OD<sub>600</sub> of bacteria reached 0.6, we induced bacteria with 1 mM, 2 mM IPTG, then cultivated for about 12 hours. Here is SDS-PAGE gel figure of VqmA<sub>phage</sub>.</p>
 +
 +
<html>
 +
<head>
 +
<meta charset="utf-8">
 +
<title>无标题文档</title>
 +
</head>
 +
<body>
 +
<center><img src="https://static.igem.wiki/teams/4645/wiki/quorum-sensing/vqma-sds.jpg" style="width:25%; "></center>
 +
<br>
 +
<center><b>Figure 2. SDS-PAGE gel figure of VqmA<sub>phage</sub>.</b> </center>
 +
<br>
 +
</body>
 +
</html>
 +
  
 
<!-- -->
 
<!-- -->
Line 12: Line 43:
 
<partinfo>BBa_K4645004 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4645004 SequenceAndFeatures</partinfo>
  
 +
===Reference===
 +
[1] Silpe JE, Bassler BL. A Host-Produced Quorum-Sensing Autoinducer Controls a Phage Lysis-Lysogeny Decision. Cell. 2019 Jan 10;176(1-2):268-280.e13.<br>
 +
[2] Duddy OP, Huang X, Silpe JE, Bassler BL. Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors. PLoS Genet. 2021 Jul 6;17(7):e1009550.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 12:26, 12 October 2023


VqmAphage
In complex with DPO, VqmAphage binds to the qtip promoter (BBa_K4645005), activating transcription. Vibrio cholerae uses a quorum-sensing (QS) system composed of the autoinducer 3,5-dimethylpyrazin-2-ol (DPO) and receptor VqmA, which together repress genes for virulence and biofilm formation [1]. VqmAVc activates expression of the vqmR gene encoding the small regulatory RNA VqmR. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates the qtip promoter (Pqtip). It’s worth noting that, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. [2]

Usage and Biology

We assembled this part with Pqtip then transfected into E. coli to sensing the density of bacteria and regulate the circuit.

What？


Figure 1. The circuit we assembled.

Characterization

Successful protein expression

We transformed vqmA into E.coli BL21(DE3) and cultivated in 37℃, 220 rpm. When the OD600 of bacteria reached 0.6, we induced bacteria with 1 mM, 2 mM IPTG, then cultivated for about 12 hours. Here is SDS-PAGE gel figure of VqmAphage.

无标题文档


Figure 2. SDS-PAGE gel figure of VqmAphage.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 535
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

[1] Silpe JE, Bassler BL. A Host-Produced Quorum-Sensing Autoinducer Controls a Phage Lysis-Lysogeny Decision. Cell. 2019 Jan 10;176(1-2):268-280.e13.
[2] Duddy OP, Huang X, Silpe JE, Bassler BL. Mechanism underlying the DNA-binding preferences of the Vibrio cholerae and vibriophage VP882 VqmA quorum-sensing receptors. PLoS Genet. 2021 Jul 6;17(7):e1009550.