Difference between revisions of "Part:BBa J23110"
 
(5 intermediate revisions by 4 users not shown)
Line 4: Line 4:
 
{{Berk2006iGEM_JCA_TCPromoters}}
 
{{Berk2006iGEM_JCA_TCPromoters}}
  
−
'''[[User:Saba|Saba]]:parts BBa_J23110 and BBa_J23118 are the same.'''
+
'''[[User:Saba|Saba]]: parts BBa_J23110 and BBa_J23118 are the same.'''
 +
 
  
−
<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
The Yale iGEM team has assembled this promoter to citrine (an improved version of YFP, with excitation peak at 514nm and emission peak at 527nm) and a T7-terminator to quantify the level of expression in E. coli and in non-model organism hosts.
 +
 +
This construct has been successfully cloned into E. coli using the broad-host range vector pKT230, a RSF1010 derived plasmid, as well as using the pPZP200 plasmid which can be transformed into agrobacterium and rhizobium. Leaky expression of citrine was observed. This construct is available as the biobrick the biobrick [https://parts.igem.org/Part:BBa_K1856003 K1856003].
 +
 +
==Characterization==
 +
[[Image:Expression_Level_for_anderson-citrine_(pKT230-Lic).jpg|300px|right]]
 +
[[Image:Expression_Level_for_constitutive_promoters_in_S_meliloti_(pPZP200-LIC).jpg‎|300px|right]]
 +
===Measured strength===
 +
The Yale iGEM team found fluorescence levels nearly fifteen times above the baseline level in E. coli cells transformed with the promoter-citrine construct. We also found this promoter to be effective in driving gene expression in S. meliloti, with fluorescence levels of over four times that of the baseline observed.
 +
 +
We further compared expression level of this promoter to two of the other promoters in the Anderson collection. The Anderson lab found relatively expression levels of 1, 0.33, and 0.10 respectively when comparing the promoters [https://parts.igem.org/Part:BBa_J23100 J23100 (Anderson Strong)], [https://parts.igem.org/Part:BBa_J23110 J23110 (Anderson Medium)] and [https://parts.igem.org/Part:BBa_J23114 J23114 (Anderson Weak)]. We found the relative expressions to be 1, 1.71 and 1.78 when we transformed our constructs into E.coli: [https://parts.igem.org/Part:BBa_K1856004 K1856004 (Anderson Strong-Citrine)], [https://parts.igem.org/Part:BBa_K1856003 K1856003 (Anderson Medium-Citrine)], [https://parts.igem.org/Part:BBa_K1856002 K1856002 (Anderson Weak-Citrine)]. This highlights that even as all three Anderson promoters are effective in driving gene expression in E. coli, the relative expression level may vary depending on the gene fused to the promoter and on the E. coli strain used. The Yale iGEM team's readings were obtained in E. coli DH10B.
 +
 +
Comparing expression levels in S. meliloti (a rhizobium strain), the relative expression values were 1, 2.36, and no significant expression for the Anderson Strong, Anderson Medium, and Anderson Weak promoters respectively.
  
 
<!-- -->
 
<!-- -->
Line 18: Line 31:
 
<partinfo>BBa_J23110 parameters</partinfo>
 
<partinfo>BBa_J23110 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
==Added by KEYSTONE_A 2020 Team==
 +
 +
J23110 can be used as constitutive promoter in bacterial cellulose producing strain [[Part:BBa_K1321306|K. rhaeticus iGEM]], whose strength is of medium level.
 +
 +
[[File:KEYSTONE A constitutive promoter strength.jpg|600px|thumb|center|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.
 +
 +
{|width='100%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
 +
<partinfo>BBa_J23100 AddReview 5</partinfo>
 +
<I>University of Texas at Austin iGEM 2019</I>
 +
|width='60%' valign='top'|
 +
 +
<h3>UT Austin iGEM 2019: Characterization of metabolic burden of the Anderson Series</h3>
 +
 +
<h4>Description</h4>
 +
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 (<partinfo>J23104</partinfo> or <partinfo>J23110</partinfo>), 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: [https://https://2019.igem.org/Team:Austin_UTexas]
 +
 +
<html>
 +
<figure>
 +
<div class = "left">
 +
<img src = "https://static.igem.org/mediawiki/parts/a/a0/AndersonCharacterization.jpg" style = "width:550px;height:500px">
 +
</div>
 +
<figcaption><b>Fig.1:</b>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.</figcaption>
 +
</figure>
 +
</html>
 +
<br><br>
 +
<html>
 +
<figure>
 +
<div class = "left">
 +
<img src = "https://static.igem.org/mediawiki/parts/8/80/T--Austin_Utexas--andersontable.png" style = "width:545px;height:375px">
 +
</div>
 +
<figcaption><b>Table.1:</b> Burden measurements for the Anderson Series promoters measured as percent reduction in growth rate ± 95% confidence interval. </figcaption>
 +
</figure>
 +
</html>
 +
 +
<h4>Importance of Characterizing Burden</h4>
 +
<p> 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.</p>

Latest revision as of 11:04, 24 October 2020

constitutive promoter family member

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

Saba: parts BBa_J23110 and BBa_J23118 are the same.


Usage and Biology

The Yale iGEM team has assembled this promoter to citrine (an improved version of YFP, with excitation peak at 514nm and emission peak at 527nm) and a T7-terminator to quantify the level of expression in E. coli and in non-model organism hosts.

This construct has been successfully cloned into E. coli using the broad-host range vector pKT230, a RSF1010 derived plasmid, as well as using the pPZP200 plasmid which can be transformed into agrobacterium and rhizobium. Leaky expression of citrine was observed. This construct is available as the biobrick the biobrick K1856003.

Characterization

Expression Level for anderson-citrine (pKT230-Lic).jpg
Expression Level for constitutive promoters in S meliloti (pPZP200-LIC).jpg

Measured strength

The Yale iGEM team found fluorescence levels nearly fifteen times above the baseline level in E. coli cells transformed with the promoter-citrine construct. We also found this promoter to be effective in driving gene expression in S. meliloti, with fluorescence levels of over four times that of the baseline observed.

We further compared expression level of this promoter to two of the other promoters in the Anderson collection. The Anderson lab found relatively expression levels of 1, 0.33, and 0.10 respectively when comparing the promoters J23100 (Anderson Strong), J23110 (Anderson Medium) and J23114 (Anderson Weak). We found the relative expressions to be 1, 1.71 and 1.78 when we transformed our constructs into E.coli: K1856004 (Anderson Strong-Citrine), K1856003 (Anderson Medium-Citrine), K1856002 (Anderson Weak-Citrine). This highlights that even as all three Anderson promoters are effective in driving gene expression in E. coli, the relative expression level may vary depending on the gene fused to the promoter and on the E. coli strain used. The Yale iGEM team's readings were obtained in E. coli DH10B.

Comparing expression levels in S. meliloti (a rhizobium strain), the relative expression values were 1, 2.36, and no significant expression for the Anderson Strong, Anderson Medium, and Anderson Weak promoters respectively.

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]


Added by KEYSTONE_A 2020 Team

J23110 can be used as constitutive promoter in bacterial cellulose producing strain K. rhaeticus iGEM, whose strength is of medium 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.