View source for Part:BBa K2319000


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<partinfo>BBa_K2319000 short</partinfo>

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).
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<img src="https://static.igem.org/mediawiki/2017/6/65/T--IISc-Bangalore--assembly-C1234-linear.png" width="100%">
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===Usage and Biology===
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<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>

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2319000 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K2319000 parameters</partinfo>
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