Difference between revisions of "Part:BBa K2319000"

 
(→‎Usage and Biology)
 
(6 intermediate revisions by the same user not shown)
Line 4: Line 4:
  
 
This part generates a fusion protein of superfolder GFP (sfGFP) and SpyCatcher (a 15.2 kDa protein) when transformed into a T7 expression system like E. coli strain BL21 (DE3).
 
This part generates a fusion protein of superfolder GFP (sfGFP) and SpyCatcher (a 15.2 kDa protein) when transformed into a T7 expression system like E. coli strain BL21 (DE3).
 +
<html>
 +
<img src="https://static.igem.org/mediawiki/2017/6/65/T--IISc-Bangalore--assembly-C1234-linear.png" width="100%">
 +
</html>
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
<html>
 +
<h2>Using this as a T7 expression backbone</h2>
 +
 +
<h3>Expressing any protein of interest</h3>
 +
 +
<p>This T7 expression backbone can be used to express any protein if its coding sequence (with a start codon) is inserted using the HindIII and NheI sites. These sites can be added to the coding sequence using PCR with primers having 5'-overhangs.</p>
 +
 +
<h3>Fusing a protein domain at the N-terminus of an existing protein</h3>
 +
 +
<p>By inserting the coding sequence of a protein domain (including the start codon) using the HindIII and AgeI sites into the T7 expression backbone (which already contains a protein coding sequence), an N-terminal fusion can be performed.</p>
 +
 +
<h2>Expressing a protein of interest</h2>
 +
 +
<p>No system is better for protein expression than E. coli BL21 (DE3) as its lon and ompT protease deficiency yields a huge amount of protein. BL21 (DE3) is a lysogenic strain that has the T7 RNA polymerase gene integrated into its genome under the lac operon; adding IPTG induces expression of T7 RNA polymerase, which recognizes the T7 promoter sequence. Any gene inserted downstream of the T7 promoter can thus be expressed.</p>
 +
 +
<p>Transform this part into BL21 (DE3) and induce protein overexpression with IPTG after the culture reaches early-log to mid-log phase (optimize this by inducing at different times).</p>
 +
 +
<h3>Our characterization</h3>
 +
 +
<p>We ran the cell lysate of the transformed clone (induced after 2 h, allowed to grow overnight at 30°C) against a protein ladder.</p>
 +
 +
<img src="https://static.igem.org/mediawiki/parts/1/15/T--IISc-Bangalore--protein.png" width="40%">
 +
 +
<p>Though the lysate shows a band just above 37 kDa, where sfGFP-SpyCatcher (~42 kDa) is expected to be, this result is not confirmatory without the WT control.</p>
 +
 +
</html>
  
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2319000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2319000 SequenceAndFeatures</partinfo>

Latest revision as of 01:48, 2 November 2017


sfGFP-SpyCatcher under T7 expression system

This part generates a fusion protein of superfolder GFP (sfGFP) and SpyCatcher (a 15.2 kDa protein) when transformed into a T7 expression system like E. coli strain BL21 (DE3).

Usage and Biology

Using this as a T7 expression backbone

Expressing any protein of interest

This T7 expression backbone can be used to express any protein if its coding sequence (with a start codon) is inserted using the HindIII and NheI sites. These sites can be added to the coding sequence using PCR with primers having 5'-overhangs.

Fusing a protein domain at the N-terminus of an existing protein

By inserting the coding sequence of a protein domain (including the start codon) using the HindIII and AgeI sites into the T7 expression backbone (which already contains a protein coding sequence), an N-terminal fusion can be performed.

Expressing a protein of interest

No system is better for protein expression than E. coli BL21 (DE3) as its lon and ompT protease deficiency yields a huge amount of protein. BL21 (DE3) is a lysogenic strain that has the T7 RNA polymerase gene integrated into its genome under the lac operon; adding IPTG induces expression of T7 RNA polymerase, which recognizes the T7 promoter sequence. Any gene inserted downstream of the T7 promoter can thus be expressed.

Transform this part into BL21 (DE3) and induce protein overexpression with IPTG after the culture reaches early-log to mid-log phase (optimize this by inducing at different times).

Our characterization

We ran the cell lysate of the transformed clone (induced after 2 h, allowed to grow overnight at 30°C) against a protein ladder.

Though the lysate shows a band just above 37 kDa, where sfGFP-SpyCatcher (~42 kDa) is expected to be, this result is not confirmatory without the WT control.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1200
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 768
    Illegal XhoI site found at 1001
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 42
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 57