Difference between revisions of "Part:BBa R0051:Experience"

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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
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== '''Experience by [http://2012.igem.org/Team:NTU-Taida 2012 iGEM NTU-Taida] ''' ==
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We tried to modify pCI, the well-known regulator from lambda phage. The original design of pCI in iGEM community is pR, which is composed of OR1 in -10 region, and OR2 in -35 regions. The transcriptional factor CI would preferentially bind to OR1 and then OR2 with cooperatively binding, via C terminal domain interaction. In the normal physiological condition, the binding of OR2 would further tighten the binding of RNA polymerase to pRM promoter and stimulate its own transcription. The binding site -10 upstream of starting point is critical to the function of the repressor. Under this notion, it seems more reasonable to pick a sequence unit with a higher affinity in -10 region. Thus we incorporate OL1, only one base pair different from OR1, and 10 times higher affinity, in our modified pCI repressor, with OR1 in -35 region.
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<center><html><img src='https://static.igem.org/mediawiki/2012/c/cb/NTU-Taida-Result-Thermal-pCI.png' width='480px'></html></center>
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== '''Experience by [https://2021.igem.org/Team:OhioState 2021 iGEM OhioState] ''' ==
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pR showed consistent activation, regardless of the presence of IPTG over the course of 24 hours, and was much more active than the control, empty reporter plasmid. This part was then used to create a composite part(BBa_K3783001) so that it would have the strength of pR but the regulatory ability of BBa_K3783000. The composite part was successful in preforming as expected. When creating composite parts with this promoter it is important to know that this part doesn't contain an RBS in it so one needs to be added.
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<center><html><img src='https://2021.igem.org/wiki/images/5/54/T--OhioState--pR-Graph.jpg' width='480px'></html></center>
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===Reference===
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# Dodd, J.B. Shearwin; Egan, J.B.; Egan, JB (2005). "Revisited gene regulation in phage lambda". ''Curr Opin Genet Dev'' 15 (2): 145–152.
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# Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor, Nucleic Acids Res. 2009 May; 37(9): 2789–2795
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== '''Experience by [https://2022.igem.wiki/ohiostate/ 2022 iGEM OhioState] ''' ==
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Inspired by OhioState 2021, we fused pR to mCherry in an attempt to determine its expression profile. Once the fusion of mCherry and pR was obtained we attempted to ligate into the pET28a backbone (given to us by Dr. David Wood's Lab). However once isolation via gel extraction was done for our backbone and inserts were digested with BamHI and NotI, ligation transformations proved fruitless after many attempts and swapping of buffers. Eventually because of time constraints were unable to pursue the problem any further.
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<center><html><img src='https://static.igem.wiki/teams/4341/wiki/engineering/gel.svg' width='480px'></html></center>
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<center><html>Figure 1. Gel Electrophoresis Result: Lane 1 rrnB P1; Lane 2 pR; Lane 3 pR-rrnB P1; Lane 4  T5-rrnB P1</html></center>
  
 
===Applications of BBa_R0051===
 
===Applications of BBa_R0051===
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<partinfo>BBa_R0051 AddReview 0</partinfo>
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<I>Shenzhen_SZMS 2016</I>
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We met the same problem Tokyo Tech has faced back in 2007 that bacteria could grow on solid/liquid medium with Amp, but nothing was found in gel after miniprep and electrophoresis. This whole procedure was repeated at least 5 times. Meanwhile, some new bacteria did grow on the Amp medium after a couple of days R0051 plasmid was transformed. We would say that for some reason R0051 plasmid gave the host bacteria resistance to Amp and then disappeared. Pretty strange and annoying.
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<partinfo>BBa_R0051 3</partinfo>
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<I>WHU-China 2012</I>
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<p>In our experiment, we used an intermediate protein cI and constructed an indirect pathway. Our device will respond to glucose, being activated at high concentration of glucose.In this way, we changed the function of the promoter R0051.
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</p>
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<p algin="justify">
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<p align="center"><img src="https://static.igem.org/mediawiki/2012/d/d5/Indirect_regulation.png" width="500"
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height="250" hspace="2" vspace="1" align="middle" /></p>
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<p align="center"> The indirect regulatory pathway</p>
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As you can see, in this pathway, protein cI will be expressed when CRP is activated at low glucose concentration.The large amount of cI repressed the promoter R0051 heavily. On the contrary, when glucose concentration is high, the promoter is derepressed.</p>
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<p align="center"><img name="" src="https://static.igem.org/mediawiki/2012/d/d5/Fluorescence_1n.png" width="520" height="409" alt=""></p>
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<p algin="justify">
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In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration, indicating that the device works as expected.In another word,the device we designed has a new function, activated by high concentration of glucose.</p>
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</html>
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Transformation and o/n culture is Okay, but we couldn't extract plasmid by miniprep. We couldn't detect band by Electrophoresis. We don't know why, but this occured 2 times.
 
Transformation and o/n culture is Okay, but we couldn't extract plasmid by miniprep. We couldn't detect band by Electrophoresis. We don't know why, but this occured 2 times.
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<partinfo>BBa_R0051 AddReview 1</partinfo>
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<I> Aberdeen_Scotland 2009 </I>
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The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (49bp).But later it was confirmed following its usage by forming the part K182002 through the PCR gel analysis and the sequencing.
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<!-- Aberdeen_Scotland 2009 user review  -->
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<partinfo>BBa_R0051 AddReview 4</partinfo>
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<I>Aberdeen_Scotland 2009 </I>
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The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (49bp).But later it was confirmed following its usage by forming the part BBa_K182002 through the PCR gel analysis and the sequencing.The plasmid miniprep also worked.
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<!-- Seu_O_China 2012 user review -->
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<I> Seu_O_China 2012</I>
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BBa_R0051 does not work well due to its rather small size. We transformed it for three times, and none of them succeed. We designed primers to PCR it out from K091230 successfully, but failed to perform the digestion since it is too small to extract from gel. It may work better if adding some nonsense sequence before the promoter.
 
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== '''Experience by [https://2021.igem.org/Team:OhioState] ''' ==
 +
 +
pR showed consistent activation, regardless of the presence of IPTG over the course of 24 hours, and was much more active than the control, empty reporter plasmid. This part was then used to create a composite part(BBa_K3783001) so that it would have the strength of pR but the regulatory ability of BBa_K3783000. The composite part was successful in preforming as expected. When creating composite parts with this promoter it is important to know that this part doesn't contain an RBS in it so one needs to be added.
 +
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<center><html><img src='https://2021.igem.org/wiki/images/5/54/T--OhioState--pR-Graph.jpg' width='480px'></html></center>

Latest revision as of 13:04, 12 October 2022

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.

Experience by [http://2012.igem.org/Team:NTU-Taida 2012 iGEM NTU-Taida]

We tried to modify pCI, the well-known regulator from lambda phage. The original design of pCI in iGEM community is pR, which is composed of OR1 in -10 region, and OR2 in -35 regions. The transcriptional factor CI would preferentially bind to OR1 and then OR2 with cooperatively binding, via C terminal domain interaction. In the normal physiological condition, the binding of OR2 would further tighten the binding of RNA polymerase to pRM promoter and stimulate its own transcription. The binding site -10 upstream of starting point is critical to the function of the repressor. Under this notion, it seems more reasonable to pick a sequence unit with a higher affinity in -10 region. Thus we incorporate OL1, only one base pair different from OR1, and 10 times higher affinity, in our modified pCI repressor, with OR1 in -35 region.


Experience by 2021 iGEM OhioState

pR showed consistent activation, regardless of the presence of IPTG over the course of 24 hours, and was much more active than the control, empty reporter plasmid. This part was then used to create a composite part(BBa_K3783001) so that it would have the strength of pR but the regulatory ability of BBa_K3783000. The composite part was successful in preforming as expected. When creating composite parts with this promoter it is important to know that this part doesn't contain an RBS in it so one needs to be added.

Reference

  1. Dodd, J.B. Shearwin; Egan, J.B.; Egan, JB (2005). "Revisited gene regulation in phage lambda". Curr Opin Genet Dev 15 (2): 145–152.
  2. Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor, Nucleic Acids Res. 2009 May; 37(9): 2789–2795

Experience by 2022 iGEM OhioState

Inspired by OhioState 2021, we fused pR to mCherry in an attempt to determine its expression profile. Once the fusion of mCherry and pR was obtained we attempted to ligate into the pET28a backbone (given to us by Dr. David Wood's Lab). However once isolation via gel extraction was done for our backbone and inserts were digested with BamHI and NotI, ligation transformations proved fruitless after many attempts and swapping of buffers. Eventually because of time constraints were unable to pursue the problem any further.

Figure 1. Gel Electrophoresis Result: Lane 1 rrnB P1; Lane 2 pR; Lane 3 pR-rrnB P1; Lane 4 T5-rrnB P1

Applications of BBa_R0051

User Reviews

UNIQe1949d2fb2a1ed94-partinfo-00000004-QINU

UNIQe1949d2fb2a1ed94-partinfo-00000005-QINU

Shenzhen_SZMS 2016

We met the same problem Tokyo Tech has faced back in 2007 that bacteria could grow on solid/liquid medium with Amp, but nothing was found in gel after miniprep and electrophoresis. This whole procedure was repeated at least 5 times. Meanwhile, some new bacteria did grow on the Amp medium after a couple of days R0051 plasmid was transformed. We would say that for some reason R0051 plasmid gave the host bacteria resistance to Amp and then disappeared. Pretty strange and annoying.

UNIQe1949d2fb2a1ed94-partinfo-00000007-QINU

BBa_R0051 3 Not understood WHU-China 2012

In our experiment, we used an intermediate protein cI and constructed an indirect pathway. Our device will respond to glucose, being activated at high concentration of glucose.In this way, we changed the function of the promoter R0051.

The indirect regulatory pathway

As you can see, in this pathway, protein cI will be expressed when CRP is activated at low glucose concentration.The large amount of cI repressed the promoter R0051 heavily. On the contrary, when glucose concentration is high, the promoter is derepressed.

In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration, indicating that the device works as expected.In another word,the device we designed has a new function, activated by high concentration of glucose.

;

UNIQe1949d2fb2a1ed94-partinfo-0000000A-QINU


•••••

Antiquity

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

•

Tokyo_Tech

Transformation and o/n culture is Okay, but we couldn't extract plasmid by miniprep. We couldn't detect band by Electrophoresis. We don't know why, but this occured 2 times.

••••

Aberdeen_Scotland 2009

The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (49bp).But later it was confirmed following its usage by forming the part BBa_K182002 through the PCR gel analysis and the sequencing.The plasmid miniprep also worked.

••

Seu_O_China 2012

BBa_R0051 does not work well due to its rather small size. We transformed it for three times, and none of them succeed. We designed primers to PCR it out from K091230 successfully, but failed to perform the digestion since it is too small to extract from gel. It may work better if adding some nonsense sequence before the promoter. UNIQe1949d2fb2a1ed94-partinfo-0000000F-QINU

Experience by [1]

pR showed consistent activation, regardless of the presence of IPTG over the course of 24 hours, and was much more active than the control, empty reporter plasmid. This part was then used to create a composite part(BBa_K3783001) so that it would have the strength of pR but the regulatory ability of BBa_K3783000. The composite part was successful in preforming as expected. When creating composite parts with this promoter it is important to know that this part doesn't contain an RBS in it so one needs to be added.