Difference between revisions of "Part:BBa P1010:Experience"

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P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.
 
P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our ''E. coli'' TOP10 cells, and keeping our ''E. coli'' DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.
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<partinfo>BBa_P1010 AddReview 4</partinfo>
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<I>iGEM Bielefeld 2010</I>
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We transformed this BioBrick into ''Escherichia coli'' JM109, DH5α, TOP10 and DB3.1. ''E. coli'' JM109 and DH5α seem to be ''ccdB'' resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in ''E. coli'' TOP10 (no colonies after transformation) and DB3.1 (lawn after transformation).
 
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Revision as of 14:26, 24 August 2010

Applications of BBa_P1010

This is the part that is present inside the BioBricks insertion site in many of the pSB series of plasmids. It is sufficient for positive selection even in pSB2* series plasmids in the absence of inducer (~4-5 copies per cell). During assembly work at the Registry, we have found that point mutations (particularly frame shifts) occur at a high enough rate that several colonies will survive during a normal transformation. -Randy

User Reviews

UNIQba135f0556f81c23-partinfo-00000000-QINU

•••••

Antiquity

This review comes from the old result system and indicates that this part worked in some test.

••••

iGEM Groningen 2009

P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our E. coli TOP10 cells, and keeping our E. coli DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.

••••

iGEM Bielefeld 2010

We transformed this BioBrick into Escherichia coli JM109, DH5α, TOP10 and DB3.1. E. coli JM109 and DH5α seem to be ccdB resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in E. coli TOP10 (no colonies after transformation) and DB3.1 (lawn after transformation).

UNIQba135f0556f81c23-partinfo-00000004-QINU