Difference between revisions of "Team:IISc-Bangalore/Design"
Raj Magesh (Talk | contribs) |
Raj Magesh (Talk | contribs) |
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<p>Our third method to induce gas vesicle aggregation (SpyCatcher-SpyTag binding) involves protein overexpression, and no system is better than <i>E. coli</i> strain BL21 (DE3)'s T7 expression system for this purpose: BL21 (DE3) is deficient in <i>lon</i> and <i>ompT</i> proteases. BL21 (DE3) 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>Our third method to induce gas vesicle aggregation (SpyCatcher-SpyTag binding) involves protein overexpression, and no system is better than <i>E. coli</i> strain BL21 (DE3)'s T7 expression system for this purpose: BL21 (DE3) is deficient in <i>lon</i> and <i>ompT</i> proteases. BL21 (DE3) 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>Using <a href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a> (T7 promoter+RBS) and | + | <p>Using <a href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a> (T7 promoter+RBS) and <a href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a> (T7 terminator), we have designed a T7 expression backbone that can be used to assemble and express fusion proteins easily.</p> |
| − | <img src="https://static.igem.org/mediawiki/2017/d/dc/T--IISc-Bangalore--assembly-BBa_K525998.png" width=" | + | <img src="https://static.igem.org/mediawiki/2017/d/dc/T--IISc-Bangalore--assembly-BBa_K525998.png" width="30%" height="30%"> |
| − | <img src="https://static.igem.org/mediawiki/2017/3/35/T--IISc-Bangalore--assembly-BBa_K731721.png" width=" | + | <img src="https://static.igem.org/mediawiki/2017/3/35/T--IISc-Bangalore--assembly-BBa_K731721.png" width="30%" height="30%"> |
<h1 id="sfgfp-spycatcher">sfGFP-SpyCatcher</h1> | <h1 id="sfgfp-spycatcher">sfGFP-SpyCatcher</h1> | ||
Revision as of 12:14, 30 October 2017
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T7 expression system
Our third method to induce gas vesicle aggregation (SpyCatcher-SpyTag binding) involves protein overexpression, and no system is better than E. coli strain BL21 (DE3)'s T7 expression system for this purpose: BL21 (DE3) is deficient in lon and ompT proteases. BL21 (DE3) 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.
Using BBa_K525998 (T7 promoter+RBS) and BBa_K731721 (T7 terminator), we have designed a T7 expression backbone that can be used to assemble and express fusion proteins easily.
sfGFP-SpyCatcher
mCherry-SpyTag
