Difference between revisions of "Part:BBa K1744000:Experience"

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Then this cassette with the right homologies is used to recombinate (through recombineering) and delete the targeted region of the plasmid/genome. After what the recombinants will be selected with ampicillin, since the gene bla is present in the cassette.
 
Then this cassette with the right homologies is used to recombinate (through recombineering) and delete the targeted region of the plasmid/genome. After what the recombinants will be selected with ampicillin, since the gene bla is present in the cassette.
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[[File:First_recomb_BBa_K1744000_1.PNG]]
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Note that during all the culture steps implicated, the cells has to be in a medium with glucose (we use 5%w/v) to keep the expression of the toxin vcrx028, that is present in the cassette, repressed.
 
Note that during all the culture steps implicated, the cells has to be in a medium with glucose (we use 5%w/v) to keep the expression of the toxin vcrx028, that is present in the cassette, repressed.
  
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The next step is to amplify both regions on each side of the targeted sequence.
 
The next step is to amplify both regions on each side of the targeted sequence.
 
Then a fusion PCR of those 2 regions has to be done.
 
Then a fusion PCR of those 2 regions has to be done.
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With the cassette obtained, another recombination is done that will delete the part BBa_K1744000 and only the cassette used to delete it will remain. To get only the recombinants of this step, the cells must be put on a medium containing arabinose (we used 1%w/v of final concentration) to trigger the killswitch in cells that wouldn't have lost the part.
 
  
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[[File:BBa_K1744000_gel_DATOX_clean_final.PNG]]
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[[File:Second_recomb_BBa_K1744000_1.PNG]]
  
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The above picture shows the screening effort used to obtain good recombinant. From our experience, it seems all recombinants after clean deletion of BBa_K1744000 are left. This means that vcrx028 can be successfully used as a highly efficient killswitch to counter select cells that did not recombine. The success rate of the method is, so far, of 100%, which is better than ampicilin since it has the tendancy to be degraded overtime.
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With the cassette obtained, as shown in the figure above, another recombination is done that will delete the part BBa_K1744000 and only the cassette used to delete it will remain. To get only the recombinants of this step, the cells must be put on a medium containing arabinose (we used 1%w/v of final concentration) to trigger the killswitch in cells that wouldn't have lost the part.
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[[File:BBa_K1744000_gel_DATOX_clean_final.PNG]]
  
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After all these steps, you will get a clean deletion in your plasmid/genome.
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The above picture shows the screening effort used to obtain good recombinant. From our experience, it seems all recombinants after clean deletion of BBa_K1744000 are good. This means that vcrx028 can be successfully used as a highly efficient killswitch to counter select cells that did not recombine. The success rate of the method is, so far, of 100%.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 23:22, 16 September 2015

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_K1744000

BBa_K1744000 is designed to achieve clean deletion in a genome or in a plasmid.

The first step is to amplify this part with appropriate primers. These have to contain the priming sites sequence indicated on each side of the part with an additional tail (~38-80bp can do) corresponding to the upstream homology to the region to delete for the forward primer and to the downstream homology for the reverse primer.

Then this cassette with the right homologies is used to recombinate (through recombineering) and delete the targeted region of the plasmid/genome. After what the recombinants will be selected with ampicillin, since the gene bla is present in the cassette.

First recomb BBa K1744000 1.PNG

Note that during all the culture steps implicated, the cells has to be in a medium with glucose (we use 5%w/v) to keep the expression of the toxin vcrx028, that is present in the cassette, repressed.

BBa K1744000 gel DATOX final 1.PNG

The above gel shows a representative success rate of BBa_K1744000. As you can see, the success rate is really high. There is no bad clones in my screening of 5 different colonies. This represent a 100% success rate. However, false positives are still possible. I would recommend to screen at least 5 clones to make sure you get at least one good clone.

The next step is to amplify both regions on each side of the targeted sequence. Then a fusion PCR of those 2 regions has to be done.

Second recomb BBa K1744000 1.PNG

With the cassette obtained, as shown in the figure above, another recombination is done that will delete the part BBa_K1744000 and only the cassette used to delete it will remain. To get only the recombinants of this step, the cells must be put on a medium containing arabinose (we used 1%w/v of final concentration) to trigger the killswitch in cells that wouldn't have lost the part.

BBa K1744000 gel DATOX clean final.PNG

The above picture shows the screening effort used to obtain good recombinant. From our experience, it seems all recombinants after clean deletion of BBa_K1744000 are good. This means that vcrx028 can be successfully used as a highly efficient killswitch to counter select cells that did not recombine. The success rate of the method is, so far, of 100%.

User Reviews

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