Difference between revisions of "Part:BBa K1433019:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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===Applications of BBa_K1433019===
 
===Applications of BBa_K1433019===
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===Single Insertion: Socket Feasibility Testing===
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<p>Posterior to verification of the function ofλ-Red system, what we aim to do is to prove that our socket site can get easily knocked-in. Since the recombinational bacterial, Socket. Coli, wasn’t prepared, socket insertion can not be strictly attested on chromosome. Therefore a surrogate assay was performed to show if our socket circuit can be well knocked-in. </p>
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[[File:ZJU_single .png|250px|thumb|center|'''Figure 1'''. The distribution of the sfGFP expression tested by flow cytometry with promoter J23110.]]
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<p>For imitating the situation where chromosome recombination occurs, we construct the designed chromosome socket circuit into a low copy number plasmid, PSB4C5, also named “Single plasmid” by us. To insert fragments into the socket circuit, we co-transform “Single” with λ-Red helper plasmid PKD46 and induce the bacteria cells with arabinose; then introduce kanamycin resistant gene segment added by “A” site and “B” site in each ends. After recombination, positive colonies are obtained and verified by PCR. Contrasting with chromosome recombination outcomes, recombination of plasmids seems to occurs easily and have higher recombination efficiency due to the higher copy number of plasmids relative to chromosome. However, PCR confirmation outcome displays that the recombination is incomplete also possibly because of plasmids’ higher copy numbers.</p>
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===User Reviews===
 
===User Reviews===

Revision as of 22:17, 17 October 2014

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Please enter how you used this part and how it worked out.

Applications of BBa_K1433019

Single Insertion: Socket Feasibility Testing

Posterior to verification of the function ofλ-Red system, what we aim to do is to prove that our socket site can get easily knocked-in. Since the recombinational bacterial, Socket. Coli, wasn’t prepared, socket insertion can not be strictly attested on chromosome. Therefore a surrogate assay was performed to show if our socket circuit can be well knocked-in.

Figure 1. The distribution of the sfGFP expression tested by flow cytometry with promoter J23110.

For imitating the situation where chromosome recombination occurs, we construct the designed chromosome socket circuit into a low copy number plasmid, PSB4C5, also named “Single plasmid” by us. To insert fragments into the socket circuit, we co-transform “Single” with λ-Red helper plasmid PKD46 and induce the bacteria cells with arabinose; then introduce kanamycin resistant gene segment added by “A” site and “B” site in each ends. After recombination, positive colonies are obtained and verified by PCR. Contrasting with chromosome recombination outcomes, recombination of plasmids seems to occurs easily and have higher recombination efficiency due to the higher copy number of plasmids relative to chromosome. However, PCR confirmation outcome displays that the recombination is incomplete also possibly because of plasmids’ higher copy numbers.


User Reviews

UNIQ9cec106b2a7e4915-partinfo-00000000-QINU UNIQ9cec106b2a7e4915-partinfo-00000001-QINU