Team:IISc-Bangalore/Design
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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.
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.
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
