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Device testing Protocol

galK::GFP Generator under induction, surface fitting using MATLAB sftool:

galK::GFP_Generator without expressing spot42 could be repressed by IPTG/aTc in an unclear way. One assumption is that the cytotoxicity of inducers impairs the capacity of expressing protein.

Comparator characterization

srlA surface fitting using MATLAB sftool:

ytfJ surface fitting using MATLAB sftool:

nanC surface fitting using MATLAB sftool:

Ratio sensor characterization
Secondary architecture of spot42 with artificial region:

Engineered Spot42 keeps repression efficiency:

Fig.components of ratio sensor is tested to show the fused sRNA keeps repression efficiency.the first figure shows that version of RpoS::spot42 keeps nearly half of repression rate. The second one is the version of orS2::spot42(mutant), which shows relatively weaker repression efficiency. The reason might be that 5 bases mutant of spot42 give a weaker pairing of spot42(mutant) and galK. But it is negligible or even better for our design.

Platform data

Fig. 1 This is aTc induction experiment of K737039. Production of spot42 is induced by aTc. The translation of galK::GFP will be inhibited by spot42 in our expectation. And experimental results show that aTc induction works well for inhibition of GFP expression which is represented with decreasing RFU.

Fig. 2 This is experimental control for IPTG induction to verify whether IPTG induction work as expected. Because of the deletion of Plac, IPTG won’t exert effect to spot42 production which means that spot42 will be constitutively expressed at relatively high levels which repress expression of GFP. To compare with Fig. 1, it is obvious that spot42 gives approximately 2-fold repression rate of galK::GFP expression.

Fig. 3 The GFP expression in all genetic circuits without any induction is shown. K737051 is lacI-repressed GFP generator. R0011-E0240 is PlacI GFP generator without tetR repression. K737038 is IPTG-inducible galK::GFP generator without IPTG induction. K737040 has been mentioned in Figure 2.

Fig.4 aTc induction experiment of BBa_K737041 is shown in (A). IPTG induction experiment of BBa_K737040 is shown in (B). Both of them serve as the experimental control for comparators and ratio sensors. Two genetic circuits when separately introduced into cells won’t respond to the inducers for LacI generator assembled with Ptet while tetR generator with Plac. The experimental results meet our expectation and pose no negative effect to GFP expression. Besides, high concentration of aTc induction to K737039 is closed to the result of (A), which indicates the effects of aTc induction and repression of spot42 to GFP expression.

Fig.5 (A) is IPTG induction experiment of K737052 which is aTc-inducible GFP generator. The genetic circuit is involved in the low copy plasmid pSB4A5 . (B) the experimental control. We can clearly to see that aTc induction of spot42 works perfectly well compared to (B).

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