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Applications of BBa_K4630102

We successfully inserted the stgRNA barcode cassette 2 fragment into the stgRNA barcode cassette 1 plasmid using the biobrick method. So we constructed stgRNA barcode cassette (1+2). After altering the conditions of different enzyme digestion and enzyme linkage, we have identified more suitable conditions for the Biobrick method. We found that 37 degree enzyme linked for 10 minutes has a higher connection success rate(Fig 1).

Figure. 1 Identification of positive colony (1+2)
(a)As shown by the arrow in the figure, using specific primers for PCR amplification, the correct connecting fragment size is 684bp. (b)Colony sequencing results

After confirming the connection between barcode Cassette 1 and barcode Cassette 2, we transformed the plasmid and pCasop or pCas into E.coli DH5alpha. Multiple positive transformants were then selected for cultivation and verification. L-arabinose and IPTG serve as dual induction signals to initiate stgRNA barcode cassette (1+2) self-targeting knockout and respond to signal recording, After inducing them, we did colony PCR. The results of colony PCR and sequencing were followed (Fig 2).

Figure. 1 Results of colony PCR and sequencing
(a)After induction, the target sequence is supposed to be truncated. The yellow reference line indicates the first-level knock-out sample and the rad reference line indicates the second-level knock-out sample. (b)(1+2) - induce -1,2,3,4,5,6,7,8 have achieved the recording results of two-level knockout. (1+2) - induce-9, 10, 11 have achieved the recording results of first level knockout. (1+2) - induce-12 is an unrecorded result.

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