Difference between revisions of "Part:BBa K103001:Experience"
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We have tested this part in conjunction with T7 polymerase and target gene (lacZ) under T7 promoter in Top10 strain. Although we have obtained white colonies in various experimental setups ([http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=7&arg0=2_June_2008&arg1=5_June_2008&arg2=6_June_2008&arg3=11_June_2008&arg4=12_June_2008&arg5=13_June_2008&arg6=16_June_2008&name=Blue/white%20and%20rifampicin%20test%20%231 #1] and [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=4&arg0=17_June_2008&arg1=18_June_2008&arg2=19_June_2008&arg3=23_June_2008&name=Blue/white%20and%20rifampicin%20test%20%232 #2]) sequencing revealed no mutations in target region (T7 promoter or lacZ ORF). We have [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=4&arg0=29_August_2008&arg1=30_August_2008&arg2=31_August_2008&arg3=1_September_2008&name=Blue-white%20screening%20and%20rifampicin%20test%20in%20GM2163 repeated] this experiment in GM2163 strain which is incapable of MutHLS mediated DNA repair due to lack of Dam and Dcm methyltransferases and obtained heritable white phenotype at high frequencies. Unfortunately we have no sequencing data to prove that mutations really occurred. | We have tested this part in conjunction with T7 polymerase and target gene (lacZ) under T7 promoter in Top10 strain. Although we have obtained white colonies in various experimental setups ([http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=7&arg0=2_June_2008&arg1=5_June_2008&arg2=6_June_2008&arg3=11_June_2008&arg4=12_June_2008&arg5=13_June_2008&arg6=16_June_2008&name=Blue/white%20and%20rifampicin%20test%20%231 #1] and [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=4&arg0=17_June_2008&arg1=18_June_2008&arg2=19_June_2008&arg3=23_June_2008&name=Blue/white%20and%20rifampicin%20test%20%232 #2]) sequencing revealed no mutations in target region (T7 promoter or lacZ ORF). We have [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=4&arg0=29_August_2008&arg1=30_August_2008&arg2=31_August_2008&arg3=1_September_2008&name=Blue-white%20screening%20and%20rifampicin%20test%20in%20GM2163 repeated] this experiment in GM2163 strain which is incapable of MutHLS mediated DNA repair due to lack of Dam and Dcm methyltransferases and obtained heritable white phenotype at high frequencies. Unfortunately we have no sequencing data to prove that mutations really occurred. | ||
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| + | ===Expression level and mutation rate of AID=== | ||
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| + | <partinfo>BBa_K103001 AddReview 5</partinfo> | ||
| + | <I>Potsdam_Bioware 2012 </I> | ||
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| + | We tried to express this RFC10 part cloned in an expression vector with arabinose promotor and a RBS. No mutation was detected on a target plasmid and no AID overexpression could be seen on an SDS gel. We assume that the distance from the RBS to the ATG start codon is suboptimal due to the distance from the XbaI site to the ATG. We suggest to clone this part as an RFC10 expression part. | ||
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Revision as of 03:22, 27 September 2012
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_K103001
Increasing intracellular mutation level
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•••••
Keton |
We have introduced aid gene to E. coli and measured level of mutations using rifampicin test. It works. The results are here:
|
Introduction of mutations to specific region of DNA
UNIQ6c11f83ec06974f6-partinfo-00000001-QINU
|
•••••
Keton |
We have tested this part in conjunction with T7 polymerase and target gene (lacZ) under T7 promoter in Top10 strain. Although we have obtained white colonies in various experimental setups ([http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=7&arg0=2_June_2008&arg1=5_June_2008&arg2=6_June_2008&arg3=11_June_2008&arg4=12_June_2008&arg5=13_June_2008&arg6=16_June_2008&name=Blue/white%20and%20rifampicin%20test%20%231 #1] and [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=4&arg0=17_June_2008&arg1=18_June_2008&arg2=19_June_2008&arg3=23_June_2008&name=Blue/white%20and%20rifampicin%20test%20%232 #2]) sequencing revealed no mutations in target region (T7 promoter or lacZ ORF). We have [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=4&arg0=29_August_2008&arg1=30_August_2008&arg2=31_August_2008&arg3=1_September_2008&name=Blue-white%20screening%20and%20rifampicin%20test%20in%20GM2163 repeated] this experiment in GM2163 strain which is incapable of MutHLS mediated DNA repair due to lack of Dam and Dcm methyltransferases and obtained heritable white phenotype at high frequencies. Unfortunately we have no sequencing data to prove that mutations really occurred. |
Expression level and mutation rate of AID
UNIQ6c11f83ec06974f6-partinfo-00000003-QINU
|
•••••
Potsdam_Bioware 2012 |
We tried to express this RFC10 part cloned in an expression vector with arabinose promotor and a RBS. No mutation was detected on a target plasmid and no AID overexpression could be seen on an SDS gel. We assume that the distance from the RBS to the ATG start codon is suboptimal due to the distance from the XbaI site to the ATG. We suggest to clone this part as an RFC10 expression part. |
UNIQ6c11f83ec06974f6-partinfo-00000005-QINU