DNA

Part:BBa_K4630100:Experience

Designed by: Zhejun Qin   Group: iGEM23_WHU-China   (2023-10-10)
Revision as of 19:26, 10 October 2023 by James Qin (Talk | contribs)


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Applications of BBa_K4630100

This part is the key element we use in the proof-of-concept of the recorder. Also, it is used to measure the basic parameter of the recording event. The overall experiments we conduct on BBa_K4630100 includes:

  1. Verification of the self-targeting and self-recombination
  2. Exploration of the concentration and induction time reliance of the inducer
  3. Exclusion of the unexpected recombination

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 certain final concentration and induce for a certain time.
  4. Spread the diluted solution to plates with appropriate antibiotics, and cultivate for 12 hours.
  5. Pick the colonies and test the editing result.

Results

Strain Construction

We successfully co-transformed our working plasmid with pCas, the vector for Cas9 and Lambda Red, into Escherichia coli DH5α (fig 1).


Fig 1 Construction of the working strain
Colony PCR results of the recorder with barcode. The white hollow arrowheads indicate the target bands. Index 5, 6, 7, 8, 9, 10, 11, 12 were successfully co-transformed.


First Concentration Matrix

We added a variable dosage of arabinose and IPTG close to the working concentration(tbl 1). Induction time for L-Arabinose is 22 hours and IPTG 5 hours. The sequencing result uncovered a successful editing (fig 2a). Large-scale screening revealed that condition 2 exhibited the highest efficiency, with efficiencies of 70%, 10%, and 38.1% for condition 2, 3, and 4, respectively (fig 2b). Non-induction controls substantiated that induction is the prerequisite for recording (fig 2c).

Table 1. The random test over inducers


User Reviews

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