Difference between revisions of "Part:BBa K627011"
 
(8 intermediate revisions by one other user not shown)
Line 1: Line 1:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K627011 short</partinfo><br>
 
<partinfo>BBa_K627011 short</partinfo><br>
−
This construct contains the cleavage site for the 14_3C protease flanked by the sequence of beta-lactamase and the TorA signal sequence for the TAT (twin arginine transporter) pathway, thus in case of the expression of the complete construct the beta lactamase can be transported into periplasm and provide ampicillin resistance. The TorA signal-sequence originate from the enzyme Trimethylamin-N-Oxid-Reduktase , which needs to be folded in the cytoplasm. The beta lactamase is an enzyme which cleaves lactam rings and makes bacteria resistant to antibiotics like ampicillin and penicillin. The cleavage site for the HRV 14 3C protease was created via oligo hybridization and ligated into the construct with cleavage site for XhoI and NheI, which makes this part modular and easy adaptable for any other protease.
 
−
 
  
 +
====Introduction====
 +
This BioBrick is a 2 part fusion of arabinose-inducible induction system and the HRV 14_3C protease.
 +
====General Information of the single sub parts====
 +
=====HRV 14_3C=====
 +
The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln/GlyPro.<br>
 +
The 22 kDa protease got its optimal activity at 4°C but also shows a high cleavage rate at 37°C. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.<br>
 +
=====Arabinose-inducible induction system=====
 +
The arabinose-inducible induction system was amplified via PCR from the pBAD_iGEMexpress vector. Based on the inhibitionary funtion of the catabolic activator protein (CAP) it got a very low expression rate without induction by arabinose. For the induction concentration between 2 mM up to 50 mM arabinose can be used to get an high expression rate.
 +
====General Function====
 +
When arabinose is added to the media (2mM up to 50 mM), the CAP lost its inhibitory function and the arabinose-inducible induction system gets activated. The protein, here the HRV 14_3C protease, after the T7 RBS gets express at a very high rate. So we can assume that this system is an effectiv protease generator.
 +
====Performance and Summary====
 +
In combination with our created ssTorA_CS-14_3C_blaFL device (BBa_K627013, [https://parts.igem.org/Part:BBa_K627013 More Information]) this protease generator was able to mediate the cell death of our transformed <i>E.coli</i> cells at ampicillin concentration up to 100 µg/ml.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
Line 12: Line 22:
  
  
−
<!-- Uncomment this to enable Functional Parameter display
+
 
−
===Functional Parameters===
+
 
 +
==Functional Parameters: Austin_UTexas==
 +
<html>
 +
<body>
 
<partinfo>BBa_K627011 parameters</partinfo>
 
<partinfo>BBa_K627011 parameters</partinfo>
−
<!-- -->
+
<h3><center>Burden Imposed by this Part:</center></h3>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.org/mediawiki/parts/f/fa/T--Austin_Utexas--no_burden_icon.png" style = "width:160px;height:120px"></center>
 +
</div>
 +
<figcaption><center><b>Burden Value: -1.4 ± 3.8% </b></center></figcaption>
 +
</figure>
 +
<p> Burden is the percent reduction in the growth rate of <i>E. coli</i> cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the
 +
<a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> pages for more information on the methods, an explanation of the sources of burden,  and other conclusions from a large-scale measurement project conducted by the <a href="http://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team</a>.</p>
 +
<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
 +
</body>
 +
</html>

Latest revision as of 22:30, 25 August 2020

Fusion part of pBAD arabinose-inducible induction system and the HRV 14_3C protease

Introduction

This BioBrick is a 2 part fusion of arabinose-inducible induction system and the HRV 14_3C protease.

General Information of the single sub parts

HRV 14_3C

The recombinant type 14_3C protease from human rhinovirus (HRV 3C) recognizes the same cleavage site as the native enzyme: LeuGluValLeuPheGln/GlyPro.
The 22 kDa protease got its optimal activity at 4°C but also shows a high cleavage rate at 37°C. The 14_3C works with a catalytic triade, containing the amino acid residues Ser-Asp-His at its active site.

Arabinose-inducible induction system

The arabinose-inducible induction system was amplified via PCR from the pBAD_iGEMexpress vector. Based on the inhibitionary funtion of the catabolic activator protein (CAP) it got a very low expression rate without induction by arabinose. For the induction concentration between 2 mM up to 50 mM arabinose can be used to get an high expression rate.

General Function

When arabinose is added to the media (2mM up to 50 mM), the CAP lost its inhibitory function and the arabinose-inducible induction system gets activated. The protein, here the HRV 14_3C protease, after the T7 RBS gets express at a very high rate. So we can assume that this system is an effectiv protease generator.

Performance and Summary

In combination with our created ssTorA_CS-14_3C_blaFL device (BBa_K627013, More Information) this protease generator was able to mediate the cell death of our transformed E.coli cells at ampicillin concentration up to 100 µg/ml. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1523
    Illegal BglII site found at 1711
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1239
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961



Functional Parameters: Austin_UTexas

BBa_K627011 parameters

Burden Imposed by this Part:

Burden Value: -1.4 ± 3.8%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.