Difference between revisions of "Part:BBa K5299205"

 
 
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<partinfo>BBa_K5299205 short</partinfo>
 
<partinfo>BBa_K5299205 short</partinfo>
  
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It consists of a promoter[1], a RBS, a sfGFP and a terminator.
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<h1 style="color:#3c6307;"><b>Description</b></h1>
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It consists of a promoter[1], a RBS, a sfGFP and a terminator. The<html> <a href="https://parts.igem.org/Part:BBa_j45992">BBa_J45992 </a> </html> is a promoter active in the stationary phase.
  
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<h1 style="color:#3c6307;"><b>Usage and Biology</b></h1>
 
It is able to produce the sfGFP according to the promoter's abilities.
 
It is able to produce the sfGFP according to the promoter's abilities.
  
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It was constructed in order to check the strength of the P3.1 promoter. In the future, it can be used by other teams that wish to utilise the P3.1 promoter, in order to estimate that P3.1 is a suitable candidate in their design for their chassis of choice.
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It was constructed in order to check the strength of the P3.1 promoter. For our experiments, this construct was used as a phase activation control for the P3.1 promoter <html> <a href="https://parts.igem.org/Part:BBa_K4583008">BBa_K4583008 </a> </html>. Also, it served as a strength control, as they are both active in the stationary phase.
  
 
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<partinfo>BBa_K5299205 parameters</partinfo>
 
<partinfo>BBa_K5299205 parameters</partinfo>
 
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<h1 style="color:#3c6307;"><b>Results</b></h1>
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Measurements for OD 600nm and fluorescence (488nm,510nm[2]) were taken over the course of 16 hours, in a 96-well microplate (black plate, clear bottoms). Clonings were done according to the Golden Braid method, leaving us with level 1 constructs in the pDGB3a1 backbone <html> <a href="https://parts.igem.org/Part:BBa_K4213058">BBa_K4213058. </a> </html> <br>
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Construct transformed into <i> E.coli  </i> BL21 (DE3) chassis, incubated at 37oC, 180rpm for 16 hours. <br>
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Plated 120 ul 5 times, out of each single liquid bacterial culture, created from the same bacterial colony, in order to establish accuracy through technical repeats. <br>
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Medium used was M9 due to minimal interference, with D-glucose serving as the carbon source. Also, served as blank, plated 5 times. <br>
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Measurements were normalised as such: using the average price of fluorescence for the 5 technical repeats and dividing it by the average price of OD. <br> Standard deviation included in the graphs. <br>
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Measurements were taken over the course of 16 hours.  Timepoint 0 is depicted as 1h, Timepoint 1 is depicted as 2h, etc. Here are the results for this part.<br>
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<html>
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<center>
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<figure>
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<img src='https://static.igem.wiki/teams/5299/mar/j23119-p3-1-j45992-b0034.png' width='700px' height='435px'
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        <thumb><center><b><small><i>Figure 1: Constructs with the BBa_J23119 or the BBa_K4583008 or the BBa_J45992 promoter,  the BBa_B0034 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. pDGB3a1 serves as a negative control. BBa_J23119 and BBa_J45992 serve as phase activation controls. BBa_J45992 also serves as strength control, due to the same phase activation.</i></small></b></center></thumb>
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</figure></center></html><br>
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===References===
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*[1]: Jaishankar, J., & Srivastava, P. (2020). Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. <i> Plasmid, 109 </i>(102491), 102491. doi:10.1016/j.plasmid.2020.102491
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*[2]: Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. <i>  Nature Biotechnology, 24 </i>(1), 79–88. doi:10.1038/nbt1172

Latest revision as of 09:54, 27 September 2024


BBa_J45992 - BBa_B0034 - BBa_I746916 - BBa_B0015

Description

It consists of a promoter[1], a RBS, a sfGFP and a terminator. The BBa_J45992 is a promoter active in the stationary phase.

Usage and Biology

It is able to produce the sfGFP according to the promoter's abilities.

It was constructed in order to check the strength of the P3.1 promoter. For our experiments, this construct was used as a phase activation control for the P3.1 promoter BBa_K4583008 . Also, it served as a strength control, as they are both active in the stationary phase.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 225


Results

Measurements for OD 600nm and fluorescence (488nm,510nm[2]) were taken over the course of 16 hours, in a 96-well microplate (black plate, clear bottoms). Clonings were done according to the Golden Braid method, leaving us with level 1 constructs in the pDGB3a1 backbone BBa_K4213058.

Construct transformed into E.coli BL21 (DE3) chassis, incubated at 37oC, 180rpm for 16 hours.

Plated 120 ul 5 times, out of each single liquid bacterial culture, created from the same bacterial colony, in order to establish accuracy through technical repeats.

Medium used was M9 due to minimal interference, with D-glucose serving as the carbon source. Also, served as blank, plated 5 times.

Measurements were normalised as such: using the average price of fluorescence for the 5 technical repeats and dividing it by the average price of OD.
Standard deviation included in the graphs.

Measurements were taken over the course of 16 hours. Timepoint 0 is depicted as 1h, Timepoint 1 is depicted as 2h, etc. Here are the results for this part.

Figure 1: Constructs with the BBa_J23119 or the BBa_K4583008 or the BBa_J45992 promoter, the BBa_B0034 RBS, the BBa_I746916 sfGFP and the BBa_B0015 terminator. pDGB3a1 serves as a negative control. BBa_J23119 and BBa_J45992 serve as phase activation controls. BBa_J45992 also serves as strength control, due to the same phase activation.

References

  • [1]: Jaishankar, J., & Srivastava, P. (2020). Strong synthetic stationary phase promoter-based gene expression system for Escherichia coli. Plasmid, 109 (102491), 102491. doi:10.1016/j.plasmid.2020.102491
  • [2]: Pédelacq, J.-D., Cabantous, S., Tran, T., Terwilliger, T. C., & Waldo, G. S. (2006). Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology, 24 (1), 79–88. doi:10.1038/nbt1172