Difference between revisions of "Part:BBa K292006:Experience"

(→‎TetR + p(TetR)+RFP (BBa_K092600) and TetR + p(TetR)+RFP+luxI (BBa_K092700))
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in the pSB1A2 backbone.  
 
in the pSB1A2 backbone.  
  
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[[File:Test_LacI2_080811_exp_wiki.jpg|thumb|LacI2 plasmid cut with EcoR1 + PstI, BglI + BclI, BglI + EcoRV, BglI + BanII]]
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[[Image:Test LacI2 080811 exp wiki.jpg|thumb|LacI2 plasmid cut with EcoR1 + PstI, BglI + BclI, BglI + EcoRV, BglI + BanII]]
  
 
Overwiev of restrctions and expected fragmentlengths:
 
Overwiev of restrctions and expected fragmentlengths:
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==== TetR + p(TetR)+RFP (BBa_K092600) and TetR + p(TetR)+RFP+luxI (BBa_K092700) ====
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At the beginning of the project our ide was to use this TetR regulated system to build the stress sensor.
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However our experience with the two biobricks is bad. Transformants did not show any signs of fluorescent
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activity, and the fragment lengths after enzyme digestion were not as expected.  
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Enter the review inofrmation here.
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<partinfo>BBa_K292006 AddReview 2</partinfo>
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<I>NTNU_Trondheim 2012</I>
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We decided to improve this part since last years team had problems with it. We ordered the biobrick from the registry, test cut it, and sequenced it. In addition we reassembled the the parts BBa_B0030, BBa_C0012 and BBa_B0014, test cut these, and sent them to sequencing.
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Both <partinfo>BBa_K822004</partinfo> and <partinfo>BBa_K292006</partinfo> was also investigated using gel electrophoresis. The gel pictures are given below:
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{|border="0"
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|[[Image:Testkutt_BBa_K292006.png|thumb]]
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|[[Image:RBS+LacI+term-gel.PNG|thumb]]
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|This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present.
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|Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense.
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When the cloning work was done, we sent both our new biobrick and the old one (<partinfo>BBa_K292006</partinfo>) to sequencing. The sequencing results can be found [http://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.
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Latest revision as of 21:32, 26 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_K292006

Reliable to use when we want to control a gene expression by using pLac promoter. In fact when LacI is not expressed, pLac is activated, and when there is an expression of LacI, pLac is inhibited. We can easily design a system to induce the transcription of LacI by using another operon as the tetracycline operon and creates a failed feedback mechanism for pLac inhibition and so, for gene expression inhibition.


User Reviews

UNIQ1dadb4f26dc94db9-partinfo-00000000-QINU



•

NTNU_Trondheim 2011

We found and orderd this part from the parts registry. Since it had RBS and double terminator it would save us from some cloning work.

When testcutting the constructs containing BBa_K292006 we got some unexpected results. Therefore we decided to digest the brick with three differnt enzymes with restriction sites in the given biobrick-sequence in addition to EcoR1 and PstI (for looking at the biobrick length) Except from the sample cut with EcorR1 and PstI, the samples were also cut with BglI wich has a recognition site in the pSB1A2 backbone.

LacI2 plasmid cut with EcoR1 + PstI, BglI + BclI, BglI + EcoRV, BglI + BanII

Overwiev of restrctions and expected fragmentlengths:

Restriction enzymes Expected lengths (bp)
EcoR1 + PstI 2053 (backbone) and 1303 (biobrick)
BglI + BclI 2032 + 1324
BglI + EcoRV 1760 + 1596
BglI + BanII 1835 + 1521

As we can see on the resulting gel none of the restriction sites inn the LacI2 fragment appear to be present. In additon the LacI2 insert looks smaller than it's supposed to be. This indicates that the biobrick sequence does not match the sequence given in the parts registry.



;

UNIQ1dadb4f26dc94db9-partinfo-00000002-QINU


UNIQ1dadb4f26dc94db9-partinfo-00000003-QINU



••

NTNU_Trondheim 2012

We decided to improve this part since last years team had problems with it. We ordered the biobrick from the registry, test cut it, and sequenced it. In addition we reassembled the the parts BBa_B0030, BBa_C0012 and BBa_B0014, test cut these, and sent them to sequencing.

Both BBa_K822004 and BBa_K292006 was also investigated using gel electrophoresis. The gel pictures are given below:

Testkutt BBa K292006.png
RBS+LacI+term-gel.PNG
This is the test cut of BBa_K292006 that the NTNU iGEM team 2011 performed. The testcut was performed with EcoRI+PstI (expected fragments: 1303 bp + 2053 bp), BglI+BclI (expected fragments: 1324 bp + 2032 bp), BglI+EcoRV (expected fragments: 1596 bp + 1760 bp) and BglI+BanII (expected fragments: 1521 bp + 1835 bp). The test cut shows that none of the expected fragments are present. Test cut of our improved part performed with NotI (first red box, expected fragments: 1276 bp + 2055 bp) and XbaI+PstI (second red box, expected fragments: 1278 bp + 2053 bp). The fragments cut with NotI makes sense on gel. In the case of cutting with XbaI+PstI, we did not expect three fragments, but the upper fragment could be uncut plasmid, since the lower fragments makes sense.

When the cloning work was done, we sent both our new biobrick and the old one (BBa_K292006) to sequencing. The sequencing results can be found [http://2012.igem.org/Team:NTNU_Trondheim/Sequencing_Improved_Construct here]. The sequencing result shows that in the old biobrick, only the terminator is present, and no LacI or RBS. In our improved biobrick, both RBS, LacI and terminator are present.

;



|}; UNIQ1dadb4f26dc94db9-partinfo-00000008-QINU