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BBa_K3245003:

BBa_K3245003（J23106-B0034-C0040-B0015-R0040-B0034-K3245008-B0014-B0015） is one of a series of composite parts designed for characterizing R0040.

Usage and biology:

This part is one of a collection of parts designed for characterizing ptetR (R0040). This part constantly expresses tetR(C0040) at middle-high level strength to inhibit the downstream ptetR from expressing sfGFP. When induced by aTc/tet, this part shows a leaping induction curve. To use this part, simply clone it into a middle/high copy plasmid vector, transfer into E.coli K-12, incubate overnight, and induce with aTc after proper dilution.

Design:

Since our project involves tetR-ptetR double plasmid expression system (Fig.1), it is essential for us to characterize ptetR by measuring the level of tetR expression required for total inhibition. To achieve this, we designed 3 different tetR-ptetR expression systems (BBa_K3245003, BBa_K3245012, BBa_K3245011), among which K3245003 expresses the highest amount of tetR (J23106-B0034).

In order not to let the promoter of antibiotics and J23106 affect the process of characterization, we inserted 2 B0015s into our expression system, one at the upstream of R0040, and the other at the downstream of sfGFP.

Characterization:

In order to characterize R0040, we cloned K3245003 into p15A, a vector with middle copy number (15-20). We then transferred p15A-K3245003 into E.coli DH10B and incubated it overnight. The culture was diluted with LB to 1/500 before induction (see team Fudan_protocol for more detail). Due to lack of access to aTc, we use tetracycline (tet) to induce R0040. Our results are shown as follows: