Difference between revisions of "Part:BBa P1010: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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how you used this part and how it worked out.
 
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===Applications of BBa_P1010===
 
===Applications of BBa_P1010===
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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).
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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===
 
===User Reviews===
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This review comes from the old result system and indicates that this part worked in some test.
 
This review comes from the old result system and indicates that this part worked in some test.
 
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|-
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<partinfo>BBa_P1010 AddReview 4</partinfo>
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<I>iGEM Groningen 2009</I>
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|width='60%' valign='top'|
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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.
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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 ''E. coli'' JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. ''E. coli'' JM109, XL1-Blue 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, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).
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<center>
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Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''.
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{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"
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|-
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!style="border-style: solid; border-width: 1px"| ''E. coli'' strain
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!style="border-style: solid; border-width: 1px"| Resistant to ''ccdB''?
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!style="border-style: solid; border-width: 1px"| Expected result?
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!style="border-style: solid; border-width: 1px"| Gyrase genotype
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|-
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|style="border-style: solid; border-width: 1px"| DB3.1
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|style="border-style: solid; border-width: 1px"| yes
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|style="border-style: solid; border-width: 1px"| yes
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|style="border-style: solid; border-width: 1px"| gyrA462
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|-
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|style="border-style: solid; border-width: 1px"| DH5α
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|style="border-style: solid; border-width: 1px"| yes
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|style="border-style: solid; border-width: 1px"| no
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|style="border-style: solid; border-width: 1px"| gyrA96
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|-
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|style="border-style: solid; border-width: 1px"| EC100D
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|style="border-style: solid; border-width: 1px"| no
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|style="border-style: solid; border-width: 1px"| yes
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|style="border-style: solid; border-width: 1px"| WT
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|-
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|style="border-style: solid; border-width: 1px"| JM109
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|style="border-style: solid; border-width: 1px"| yes
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|style="border-style: solid; border-width: 1px"| no
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|style="border-style: solid; border-width: 1px"| gyrA96
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|-
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|style="border-style: solid; border-width: 1px"| TOP10
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|style="border-style: solid; border-width: 1px"| no
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|style="border-style: solid; border-width: 1px"| yes
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|style="border-style: solid; border-width: 1px"| WT
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|-
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|style="border-style: solid; border-width: 1px"| XL1-Blue
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|style="border-style: solid; border-width: 1px"| yes
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|style="border-style: solid; border-width: 1px"| no
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|style="border-style: solid; border-width: 1px"| gyrA96
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|-
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|}
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</center>
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It seems that not only the gyrase mutation gyrA462 is causing a ''ccdB'' resistance. Also the gyrase mutation gyrA96 gives ''E. coli'' a ''ccdB'' resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.
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|}
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{|width='80%' style='border:1px solid gray'
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|-
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|width='10%'|
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<partinfo>BBa_P1010 AddReview 5</partinfo>
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<I>iGEM Bielefeld 2011</I>
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|width='60%' valign='top'|
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P1010 works as expected in ''E. coli'' KRX, BL21(DE3) and MACH1.
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|};
 
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<!-- DON'T DELETE --><partinfo>BBa_P1010 EndReviews</partinfo>

Latest revision as of 17:45, 15 May 2011

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

UNIQa5d30431450bc812-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 E. coli JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. E. coli JM109, XL1-Blue 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, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).


Results of the transformation of the cell-death gene ccdB, BioBrick BBa_P1010, into different strains of E. coli.

E. coli strain Resistant to ccdB? Expected result? Gyrase genotype
DB3.1 yes yes gyrA462
DH5α yes no gyrA96
EC100D no yes WT
JM109 yes no gyrA96
TOP10 no yes WT
XL1-Blue yes no gyrA96


It seems that not only the gyrase mutation gyrA462 is causing a ccdB resistance. Also the gyrase mutation gyrA96 gives E. coli a ccdB resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.

•••••

iGEM Bielefeld 2011

P1010 works as expected in E. coli KRX, BL21(DE3) and MACH1.

;

UNIQa5d30431450bc812-partinfo-00000006-QINU