Difference between revisions of "Part:BBa K4630113:Experience"

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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.
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The knock-out of the first cassettes were successful(fig 1a). However, the second cassette remained intact. In line with the previous data from stgRNA-cassette 1, we realized that there might be some technical obstacles of the knock-out of the last cassette of the device. Comparing with stgRNA-barcode-cassette 1, we have more confidence that the homologous arms play a important role. (see also documentations of BBa_K4630113[https://parts.igem.org/Part:BBa_K4630110]) .
  
 
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Fig 1 The induction knock-out results of stgRNA-cassette (1+2) and stgRNA-cassette 1<br>
 
Fig 1 The induction knock-out results of stgRNA-cassette (1+2) and stgRNA-cassette 1<br>
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(a) Induction knock-out result of stgRNA-cassette 1, All of them remained intact.<br>
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(a) Induction knock-out result of stgRNA-cassette (1+2).<br>
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(b) Induction knock-out results of stgRNA-barcode-cassette 1. <br>
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(b)Induction knock-out result of stgRNA-cassette 1, All of them remained intact.
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(c) Induction knock-out  result of stgRNA-cassette (1+2) . <br>
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Revision as of 00:58, 12 October 2023


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K4630113

This part is integrated and contains two cassettes. As the intermediate of the single cassette and multiple cassettes, BBa_K4630113 functions as the trouble shooting tool and helps the primary proof-of-concept of the cascade system.

Experiments

The experiment we carried out on BBa_K4630113 is mainly the induction and verification.


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

The knock-out of the first cassettes were successful(fig 1a). However, the second cassette remained intact. In line with the previous data from stgRNA-cassette 1, we realized that there might be some technical obstacles of the knock-out of the last cassette of the device. Comparing with stgRNA-barcode-cassette 1, we have more confidence that the homologous arms play a important role. (see also documentations of BBa_K4630113[1]) .

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+2).
(b)Induction knock-out result of stgRNA-cassette 1, All of them remained intact.

User Reviews

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