Difference between revisions of "Part:BBa J23117"

Latest revision as of 11:22, 27 October 2020

constitutive promoter family member

 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

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

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]

This promoter was BioBricked in 2015 competition with a PAM rich Upstream regulation sequence (URS), BBa_K1723001. This PAM rich sequence is needed for the protein dCas9 to bind to the promoter and act as a programmable transcription regulator . Another promoter, PAM rich URS J23117Alt ( BBa_K1723005 ) was created by mutating sequences of the improved J23117 promoter keeping intact -10 sequence and -35 sequence to keep a similar promoter strength.

Added by KEYSTONE_A 2020 Team

J23117 can be used as a constitutive promoter in bacterial cellulose producing strain K. rhaeticus iGEM, but it is of relatively low expression level.

Visual Results as Normally Open Switches

Figure above: Constitutive promoter average strengths in K. rhaeticus iGEM and E. coli, normalized against J23104. Although all promoters are functional, their relative strengths differ between K. rhaeticus and E. coli. For K. rhaeticus, data is shown as grey bars, with standard deviation of N=3 biological replicates, characterized in liquid HS-medium containing cellulase, measured 3 h post-inoculation. Relative promoter strengths in E. coli are superimposed as black stripes.

References: Florea, M., Hagemann, H., Santosa, G., Abbott, J., Micklem, C. N., Spencer-Milnes, X., ... & Chughtai, H. (2016). Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proceedings of the National Academy of Sciences, 113(24), E3431-E3440.

•••••

University of Texas at Austin iGEM 2019

UT Austin iGEM 2019: Characterization of metabolic burden of the Anderson Series

Description

The 2019 UT Austin iGEM team transformed the Anderson Series promoters into our 'burden monitor' DH10B strain of E. coli, which contains a constitutive GFP cassette in the genome of the cell. GFP expression fluctuates depending on the number of ribosomes available. Using this strain, we characterized the relative burden (percent reduction in growth rate) of each Anderson Series part. Our results showed a range of growth rate reductions for each of these parts due to ribosomal reallocation from the genome of the host cell, towards the expression of RFP. Anderson Series parts with strong promoters are depicted with darker red colors and Anderson Series parts with weak promoters are depicted with lighter pink colors to show relative RFP expression. We saw a positive correlation between relative promoter strength and metabolic burden; parts with stronger promoters expressed less GFP and had a lower growth rate than parts with weaker promoters. The regression line for the graph below was constructed by measuring the burden of 5 parts that were created by the 2019 UT Austin iGEM team that each contained an Anderson Series promoter (BBa_J23104 or BBa_J23110), an RBS of varying strength, and a BFP reporter. For more information on characterization of these parts through the burden monitor, visit our team’s wiki page: [1]

**Fig.1:**Growth vs GFP Expression graph showing the relative burden positions of the Anderson Series promoters. The parts with strong promoters are depicted in dark red and are clustered near the bottom of the graph because they have lower growth rates and express lower levels of GFP as a result of high cellular burden. The parts with weaker promoter are depicted in light pink ad are clustered near the top of the graph because they have higher growth rates and express higher levels of GFP as a result of low cellular burden.

**Table.1:** Burden measurements for the Anderson Series promoters measured as percent reduction in growth rate ± 95% confidence interval.

Importance of Characterizing Burden

Although often we cannot avoid using a specific burdensome part, knowing in advance that it is burdensome, and that it has a high chance of mutating into a non-functional genetic device, can help with troubleshooting and coming up with alternatives. In the specific case of fluorescent protein-expressing devices, Fluorescence-activated cell sorting (FACS) can be used to filter out individual cells that meet a certain fluorescence threshold. This way, the cells expressing lower levels of the fluorescent protein are weeded out of the population.

Added by SJTU-BioX-Shanghai 2020 Team

J23117 is a weak promoter and it can lead to a quite low expression level in the absence of dCas9-ω. So, it is a good choice to be designed as a part of CRISPRa system. It will cause a significant difference in expression level of reporter after dCas9 binding.

Figure above : Quoted from a study by Broad Institute

W103 and W108 in the figure means different ways to put the target site for dCas9-ωupstream of the promoter. Comparing the dCas9-ω activation of GFP expression from the three constructs using different gRNA, J23117 performs best when induced by the dCas9-ω both in W103 and in W108.

References:

[1]David Bikard,Wenyan Jiang,Poulami Samai,Ann Hochschild,Feng Zhang,Luciano A. Marraffini1.Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system[J]. Nucleic Acids Research,2013,41(15):7429-7437.

@@ Line 67: / Line 67: @@
 ==Added by SJTU-BioX-Shanghai 2020 Team==
-As J23117 is a weak promoter, it can be a good choice when design a part of CRISPRa system. Because it has a quite low expression level in the absence of dCas9-ω, and has a normal expression level when dCas9-ω binding to the on-target site or off-target site, which means it will make a more significant difference in expression of report before and after dCas9 binding.
+J23117 is a weak promoter and it can lead to a quite low expression level in the absence of dCas9-ω. So, it is a good choice to be designed as a part of CRISPRa system.  It will cause a significant difference in expression level of reporter after dCas9 binding.
 [[File:Quoted_from_a_study_by_Broad_Institute.png]]
@@ Line 73: / Line 73: @@
 '''Figure above : Quoted from a study by Broad Institute'''
-W103 and W108 in the figure means different ways to put the target site for dCas9-ωupstream of the promoter. Comparing the dCas9-ω activation of gfp expression from the three constructs using different gRNA, J23117 could be best induced by the targeting of dCas9-o to both binding sites both in W103 and in W108.
+W103 and W108 in the figure means different ways to put the target site for dCas9-ωupstream of the promoter. Comparing the dCas9-ω activation of GFP expression from the three constructs using different gRNA, J23117 performs best when induced by the dCas9-ω both  in W103 and in W108.
 '''References:'''
 [1]David Bikard,Wenyan Jiang,Poulami Samai,Ann Hochschild,Feng Zhang,Luciano A. Marraffini1.Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system[J]. Nucleic Acids Research,2013,41(15):7429-7437.