Difference between revisions of "Part:BBa J23108"
 
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For video explanation on this promoter family and its use, see [http://www.synbiotrails.org/#!/trail?id=org.clothocad.trails.youtube.TranscriptionModels this].
 
For video explanation on this promoter family and its use, see [http://www.synbiotrails.org/#!/trail?id=org.clothocad.trails.youtube.TranscriptionModels this].
 
{{Berk2006iGEM_JCA_TCPromoters}}
 
{{Berk2006iGEM_JCA_TCPromoters}}
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<!--[[2017-OUC-China]]-->
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<strong>OUC-China</strong> developed part BBa_K2314212 from this part.
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5'UTR will be transcribed and can be used as a regulatory element to adjust the
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translation process. We chose J23108 and added the 5'UTR sequence (UTRrpsT)
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thereafter. We found that 6h fluorescence intensity increased by more than 1.5-
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fold. For more information about our new part, please click here:https://parts.igem.org/Part:BBa_K2314212
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[[Image:T--OUC-China--improve.illustration.png|500px|thumb|center|UTRrpsT enhances protein expression by structure at the 5’UTR.]]
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[[Image:T--OUC-China-utrrbs.png|500px|thumb|center|The enhancement of UTRrpsT may be related to its secondary structure, as shown by the website (http://rna.tbi.univie.ac.at/).]]
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[[Image:T--OUC-China--FI.png|500px|thumb|center|FI／Abs600： J23106 vs J23106+UTR]]
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<br>
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<br>
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<br>
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<strong>Manchester 2017</strong> used this part to create part MediumPromoter_PduD(1-20)_mCherry (BBa_K2213007). This promoter was combined with PduD(1-20) to create a tag with medium expression levels. The mCherry tagged PduD(1-20) localisation tag displayed higher fluorescence levels under the medium promoter as compared to under low strength (BBa_K2213006) promoter, and similar fluorescence levels to the high strength (BBa_K2213008) promoter. This result was unexpected and should be taken into consideration when choosing promoters.<br>
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<br>
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More information can be found here: https://parts.igem.org/Part:BBa_K2213007<br>
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<br>
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https://static.igem.org/mediawiki/2017/3/31/TagExpression500p.jpg<br>
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<br>
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==Characterization: Jilin_China 2019==
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<h3>'''Group:'''</h3> Jilin_China 2019
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<h5>
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<P style="text-indent:2em;">
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We hope that when we use an unrepresentated promoter, we can easily and accurately get its relative strength in the already characterized promoter family.
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</P>
 +
 +
<P style="text-indent:2em;">
 +
Therefore, we constructed the promoter strength measurement vector BBa_K3078100 and characterized the promoter with a large intensity gradient of the Anderson promoter family, BBa_J23119, BBa_J23104, BBa_J23108, BBa_J23105, BBa_J23114, and it is expected to establish a standard curve with fluorescence intensity.
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</p>
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</h5>
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[[File:表征1.png|600px|center|表征1]]
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<center style="text-align:left;">
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Figure 1. The fluorescence of each promoter changes with time (fluorescence / OD600). Different elements are connected to the same measurement carrier, cultured overnight, diluted to OD = 0.02 the next day, and the fluorescence at absorption wavelength 528nm was measured under excitation wavelength 485nm light.
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</center>
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<center style="text-align:left;">
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Table 1. Reported relative expression and corrected relative expression. We calculated the relative fluorescence intensity of each promoter for it based on J23119.
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</center>
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[[File:表征2.png|600px|center|表征2]]
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<h5>
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<P style="text-indent:2em;">
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The fluorescence data showed the intensity of BBa_J23104 and BBa_J23119 is much too high to constitute a linear relationship with the other three promoters. The reason of which might be the fusion genes influenced the relatve expression level of the promoter.
 +
</P>
 +
 +
<P style="text-indent:2em;">
 +
We invited JiangnanU_China to repeat, and the result of its characterization was not consistent with reported relative expression .Therefore, we speculated that the relative strength might be affected by experimental strains, plasmids and other factors. Under our experimental conditions, the relative strength of Anderson promoter was not consistent with reported relative expression.
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</p>
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</h5>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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<!-- Uncomment this to enable Functional Parameter display
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−
===Functional Parameters===
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==Functional Parameters: Austin_UTexas==
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<html>
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<body>
 
<partinfo>BBa_J23108 parameters</partinfo>
 
<partinfo>BBa_J23108 parameters</partinfo>
−
<!-- -->
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<h3><center>Burden Imposed by this Part:</center></h3>
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<figure>
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<div class = "center">
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<center><img src = "https://static.igem.org/mediawiki/parts/f/fa/T--Austin_Utexas--no_burden_icon.png" style = "width:160px;height:120px"></center>
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</div>
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<figcaption><center><b>Burden Value: 0.0 ± 1.5% </b></center></figcaption>
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</figure>
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<p> Burden is the percent reduction in the growth rate of <i>E. coli</i> cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the
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<a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> pages for more information on the methods, an explanation of the sources of burden,  and other conclusions from a large-scale measurement project conducted by the <a href="http://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team</a>.</p>
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<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
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</body>
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</html>

Latest revision as of 03:02, 26 August 2020

constitutive promoter family member For video explanation on this promoter family and its use, see [http://www.synbiotrails.org/#!/trail?id=org.clothocad.trails.youtube.TranscriptionModels this].

BerkiGEM2006-PromotersEppendorfs.jpg
BerkiGEM2006-Promoters.jpg

 
 Variant RFP (au)
 J23112           1
 J23103           17
 J23113           21
 J23109           106
 J23117           162
 J23114           256
 J23115           387
 J23116           396
 J23105           623
 J23110           844
 J23107           908
 J23106           1185
 J23108           1303
 J23118           1429
 J23111           1487
 J23101           1791
 J23104           1831
 J23102           2179
 J23100           2547
PBca1020-r0040.jpg

Constitutive promoter family
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. J23119 is the "consensus" promoter sequence and the strongest member of the family. All parts except J23119 are present in plasmid J61002. Part J23119 is present in pSB1A2. This places the RFP downstream of the promoter. Reported activities of the promoters are given as the relative fluorescence of these plasmids in strain TG1 grown in LB media to saturation. See part BBa_J61002 for details on their use.

These promoter parts can be used to tune the expression level of constitutively expressed parts. The NheI and AvrII restriction sites present within these promoter parts make them a scaffold for further modification. JCAraw


OUC-China developed part BBa_K2314212 from this part. 5'UTR will be transcribed and can be used as a regulatory element to adjust the translation process. We chose J23108 and added the 5'UTR sequence (UTRrpsT) thereafter. We found that 6h fluorescence intensity increased by more than 1.5- fold. For more information about our new part, please click here:https://parts.igem.org/Part:BBa_K2314212

UTRrpsT enhances protein expression by structure at the 5’UTR.
The enhancement of UTRrpsT may be related to its secondary structure, as shown by the website (http://rna.tbi.univie.ac.at/).
FI／Abs600： J23106 vs J23106+UTR




Manchester 2017 used this part to create part MediumPromoter_PduD(1-20)_mCherry (BBa_K2213007). This promoter was combined with PduD(1-20) to create a tag with medium expression levels. The mCherry tagged PduD(1-20) localisation tag displayed higher fluorescence levels under the medium promoter as compared to under low strength (BBa_K2213006) promoter, and similar fluorescence levels to the high strength (BBa_K2213008) promoter. This result was unexpected and should be taken into consideration when choosing promoters.

More information can be found here: https://parts.igem.org/Part:BBa_K2213007

TagExpression500p.jpg

Characterization: Jilin_China 2019

Group:

Jilin_China 2019

We hope that when we use an unrepresentated promoter, we can easily and accurately get its relative strength in the already characterized promoter family.

Therefore, we constructed the promoter strength measurement vector BBa_K3078100 and characterized the promoter with a large intensity gradient of the Anderson promoter family, BBa_J23119, BBa_J23104, BBa_J23108, BBa_J23105, BBa_J23114, and it is expected to establish a standard curve with fluorescence intensity.

表征1

Figure 1. The fluorescence of each promoter changes with time (fluorescence / OD600). Different elements are connected to the same measurement carrier, cultured overnight, diluted to OD = 0.02 the next day, and the fluorescence at absorption wavelength 528nm was measured under excitation wavelength 485nm light.


Table 1. Reported relative expression and corrected relative expression. We calculated the relative fluorescence intensity of each promoter for it based on J23119.

表征2

The fluorescence data showed the intensity of BBa_J23104 and BBa_J23119 is much too high to constitute a linear relationship with the other three promoters. The reason of which might be the fusion genes influenced the relatve expression level of the promoter.

We invited JiangnanU_China to repeat, and the result of its characterization was not consistent with reported relative expression .Therefore, we speculated that the relative strength might be affected by experimental strains, plasmids and other factors. Under our experimental conditions, the relative strength of Anderson promoter was not consistent with reported relative expression.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Functional Parameters: Austin_UTexas

BBa_J23108 parameters

Burden Imposed by this Part:

Burden Value: 0.0 ± 1.5%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.