Part:BBa_K4630110:Experience

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Applications of BBa_K4630110

As The building block of the cascade system, we carried out subcloning and verification on this part.

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

Using different induction conditions (tbl 1), we tested the function of a single cassette. However, the target sequences remained all intact (fig 1a). Interestingly, the stgRNA-barcode-cassette 1 got positive result (fig 1b), and the positive results of stgRNA-cassette (1+2) indicates that stgRNA 1 is functional (fig 1c). Comparing the two part series, we ascribe to the position of the homologous arm: the homologous arm was set downstream of each cassette in stgRNA-barcode-cassette 1 , while the upstream of the next cassette in stgRNA-cassette 1. As a result, the single cassette we have tested has no inherent homologous arm.

Table1 The induction group

Fig 1 The induction knock-out results of stgRNA-cassette (1+2) and stgRNA-cassette 1
(a) Induction knock-out result of stgRNA-cassette 1, All of them remained intact.
(b) Induction knock-out results of stgRNA-barcode-cassette 1.
(c) Induction knock-out result of stgRNA-cassette (1+2) .

User Reviews

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