Part:BBa_K4630114:Experience

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Applications of BBa_K4630114

This part is the more integrated form that contains three cassettes. Using this part, we can verify the concept of multi-level recording and gain comprehensive data.

General Induction Protocol

1. Pick a colony from the verified plate, cultivate in 5ml liquid LB media for 12 hours.
2. Transfer 100µl bacteria solution to 3ml new liquid LB media. Cultivate to an OD600 of 0.3~0.5.
3. Add L-Arabinose and IPTG to final concentrations of 2g/L and 3g/L, and induce for 22 hours and 5 hours, respectively.
4. Spread the diluted solution to plates with appropriate antibiotics and cultivate for 12 hours.
5. Pick the colonies and test the editing result.

Result

Proof-of-Concept

We implement induction over the part, and it is successfully knock-out.

Fig 1 The successful knock-out of the stgRNA-cassette (1+2+3)

Time-Gradient Test

Also, according to our PI's advice, IPTG induction do harm to the cells. However, refractory phase is a key parameter of the multi-level recorder. So, we performed time-gradient tests over the part (tbl 1).

All of the samples achieve full knock-out, at a rate of 100% (fig 2). However, the time-gradient within 2 hours did not really set up, because of inconsistency with the time-gradient tes in BBa_K4630100. Not surprisingly, the test carried out on stgRNA-cassette (1+2+3) showed some kind of leakage expression. More experiments of different promoters should be taken.

Table 1 Time-gradient test

Fig 2 Time-gradient test over the part

User Reviews

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