Difference between revisions of "Team:IISc-Bangalore/Design"
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| − | <h1 id="t7-expression-system">T7 expression system</h1> | + | <h1 id="t7-expression-system">Designing a T7 expression system</h1> |
<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> | ||
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<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/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="30%" height="30%"> | <img src="https://static.igem.org/mediawiki/2017/3/35/T--IISc-Bangalore--assembly-BBa_K731721.png" width="30%" height="30%"> | ||
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| + | <h2>Choice of BioBricks</h2> | ||
<p><a href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a> (T7 promoter+RBS) was chosen since a strong RBS <a href="http://parts.igem.org/Part:BBa_B0034">B0034</a> is used, and this allows for maximal expression of our proteins of interest. <a href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a> (T7 terminator) was chosen instead of the standard <a href="">B0015</a> double terminator as its <i>in vivo</i> termination efficiency is greater, as characterized by <a href="http://parts.igem.org/Part:BBa_K731700">BBa_K731700</a>.</p> | <p><a href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a> (T7 promoter+RBS) was chosen since a strong RBS <a href="http://parts.igem.org/Part:BBa_B0034">B0034</a> is used, and this allows for maximal expression of our proteins of interest. <a href="http://parts.igem.org/Part:BBa_K731721">BBa_K731721</a> (T7 terminator) was chosen instead of the standard <a href="">B0015</a> double terminator as its <i>in vivo</i> termination efficiency is greater, as characterized by <a href="http://parts.igem.org/Part:BBa_K731700">BBa_K731700</a>.</p> | ||
| − | <p>A HindIII restriction site, a start codon and an AgeI restriction site (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2319001">BBa_K2319001</a>) are added immediately downstream of the T7 promoter+RBS. A number of design considerations went into this choice of restriction sites</p> | + | <h2>Modification — <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2319001">BBa_K2319001</a> (HindIII+ATG+AgeI scar)</h2> |
| + | <p>A HindIII restriction site, a start codon and an AgeI restriction site (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2319001">BBa_K2319001</a>) are added immediately downstream of the T7 promoter+RBS. A number of design considerations went into this choice of restriction sites</p>. | ||
<h1 id="sfgfp-spycatcher">sfGFP-SpyCatcher</h1> | <h1 id="sfgfp-spycatcher">sfGFP-SpyCatcher</h1> | ||
Revision as of 12:36, 30 October 2017
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Designing a 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.
Choice of BioBricks
BBa_K525998 (T7 promoter+RBS) was chosen since a strong RBS B0034 is used, and this allows for maximal expression of our proteins of interest. BBa_K731721 (T7 terminator) was chosen instead of the standard B0015 double terminator as its in vivo termination efficiency is greater, as characterized by BBa_K731700.
Modification — BBa_K2319001 (HindIII+ATG+AgeI scar)
A HindIII restriction site, a start codon and an AgeI restriction site (BBa_K2319001) are added immediately downstream of the T7 promoter+RBS. A number of design considerations went into this choice of restriction sites
.sfGFP-SpyCatcher
mCherry-SpyTag
