Difference between revisions of "Part:BBa K103001:Experience"

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<I>Potsdam_Bioware 2012 </I>
 
<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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'''In chinese hamster ovary (CHO) cells''': Strong Expression (with an additional CMV-promoter and hGH polyadenylation sequence in pSB1C3-[https://parts.igem.org/Part:BBa_K929000 BBa_K929000]) works and an increased mutation rate was measured. <br>
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'''In ''E. coli ER2738'' '''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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Latest revision as of 17:34, 29 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

•••••

Keton

We have introduced aid gene to E. coli and measured level of mutations using rifampicin test. It works. The results are here:

  • [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=3&arg0=19_May_2008&arg1=20_May_2008&arg2=21_May_2008&name=Rifampicin%20test%20%231 Rifampicin test #1]
  • [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=3&arg0=22_May_2008&arg1=23_May_2008&arg2=24_May_2008&name=Rifampicin%20test%20%232 Rifampicin test #2]
  • [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=3&arg0=28_May_2008&arg1=29_May_2008&arg2=2_June_2008&name=Rifampicin%20test%20%233 Rifampicin test #3]

Introduction of mutations to specific region of DNA

UNIQ85e329c561f5d42f-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.


Improvements of this part are available

•••••

Potsdam_Bioware 2012

We have developed several improved parts for usage in our this year [http://2012.igem.org/Team:Potsdam_Bioware project] based on BBa K103001:

BBa_K929000 AID with CMV promoter and hGH-polyadenylation signal sequence

CMV-promoter & hGH polyadenylationsequence were added to BBa_K103001 for strong expression.

BBa_K929001 modified_AID without NES, with NLS and Kozak sequence

To improve the mutation rate the AID was located into the nucleus, because there it mutates transcribed DNA. Therefor an aditional NLS(Nuclear Localization Sequence)was added and the naturally occurring NES(Nuclear Exprot Sequence) was removed.For stronger Expression an Kozak Sequence was added. To fuse modified AID((C-terminal of mod. AID) with RFC 25 parts, we added an AgeI restriction site.

BBa_K929002 modified_AID with CMV and hGH-polyA

For strong expression of the previously described part an CMV-promoter and hGH polyadenylation sequence were added.

BBa_K929004 modified_AID+eGFP

The modified AID was fused with eGFP to check 1.)the tranfection success 2.)the cellular lokalization of the improved AID and 3.) to select transfected cells via FACS.

BBa_K929003modified AID with CMV, hGH-polyA and eGFP

For strong expression of the previously described part an CMV-promoter and hGH polyadenylation sequence were added.

Click the BioBrick numbers to see experience with these improved parts.

Expression level and mutation rate of AID

UNIQ85e329c561f5d42f-partinfo-00000004-QINU

•••••

Potsdam_Bioware 2012

In chinese hamster ovary (CHO) cells: Strong Expression (with an additional CMV-promoter and hGH polyadenylation sequence in pSB1C3-BBa_K929000) works and an increased mutation rate was measured.
In E. coli ER2738 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.


UNIQ85e329c561f5d42f-partinfo-00000006-QINU