Difference between revisions of "Part:BBa K1847008"
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We wanted to know how the promoter would respond to the lactate presence and how important is the architecture of the promoter in this response. For this regard, we built several promoters. [[Part:BBa_K1847002]], [[Part:BBa_K1847003]], [[Part:BBa_K1847004]], [[Part:BBa_K1847005]], [[Part:BBa_K1847006]], [[Part:BBa_K1847007]], [[Part:BBa_K1847008]], and [[Part:BBa_K1847009]].
 
We wanted to know how the promoter would respond to the lactate presence and how important is the architecture of the promoter in this response. For this regard, we built several promoters. [[Part:BBa_K1847002]], [[Part:BBa_K1847003]], [[Part:BBa_K1847004]], [[Part:BBa_K1847005]], [[Part:BBa_K1847006]], [[Part:BBa_K1847007]], [[Part:BBa_K1847008]], and [[Part:BBa_K1847009]].
  
−
<h2>Experimental Set-Up</h2>
+
<h3>Genetic design for characterization with LldR</h3>
−
<h3>Plasmids</h3>
+
To test our promoter we designed a plasmid containing the promoter with sfGFP in a medium copy plasmid pSEVA261 and transformed it into <i>Escherichia coli</i> TOP10. Then, we added a second plasmid containing lldR with a medium strong promoter ([[Part:BBa_J23118]]) and a strong RBS ([[Part:BBa_B0034]]) in pSEVA371 backbone. According to literature, LldR will repress the promoter and by the addition of lactate the production of GFP will start, as the repressor will be removed.  
−
To test our promoter we designed a plasmid containing the promoter with sfGFP in a medium copy plasmid pSEVA261 and transformed it into <i>Escherichia coli</i> TOP10. Then, we added a second plasmid containing lldR with a medium strong promoter (BBa_J23118) and a strong RBS (BBa_B0034) in pSEVA371 backbone. We expect LldR to repress the promoter and by the addition of lactate the production of GFP will start, as the repressor will be removed.
+
<h3>Genetic design for characterization with LldP-LldP</h3>
−
We tried another set-up in which we added LldP into our LldR plasmid. LldP is an L-lactate permease which import L-lactate into the cell.  
+
The second step for our characterization was to add an L-lactate permease (LldP), which despite its name is an active transporter of L-lactate, D-lactate and glycolate. To understand the effect of LldP (L-lactatep permease) in our system, we tried the following conditions:
−
In summary, we tried our system in three different conditions:
+
 
<ul>
 
<ul>
−
   <li>Plasmid with the promoter and sfGFP in pSEVA261 + J23118-B0034-lldR in pSEVA371</li>
+
   <li>Plasmid with the promoter and sfGFP in medium copy number plasmid+ J23114-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in low copy number plasmid</li>
−
  <li>Plasmid with the promoter and sfGFP in pSEVA261 + J23114-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in pSEVA371</li>
+
   <li>Plasmid with the promoter and sfGFP in medium copy number plasmid + J23118-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in low copy number plasmid</li>
−
   <li>Plasmid with the promoter and sfGFP in pSEVA261 + J23118-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in pSEVA371</li>
+
</ul>  
−
</ul>
+
  
 
<h3>Plate reader</h3>
 
<h3>Plate reader</h3>
Line 40: Line 38:
 
     <li>n: Hill coefficient (a representation of cooperativity), unitless</li>
 
     <li>n: Hill coefficient (a representation of cooperativity), unitless</li>
 
</ul>
 
</ul>
 +
  
 
{|class="wikitable"
 
{|class="wikitable"
Line 46: Line 45:
 
|-
 
|-
 
! rowspan="4" scope="row" | Coefficient values  
 
! rowspan="4" scope="row" | Coefficient values  
−
| K=1075 (889.5, 1261) || K=1751 (918.5, 2584) || K=337.7 (152.4, 522.9)
+
| K=697.7 (75.16, 1320) || K=1977 (474.5, 3479) || K=1337 (470, 2203)
 
|-
 
|-
−
| al = 479.3 (-142.2, 1101) ||al = 550 (-1635, 2735)||al = 3444 (2571, 4317)
+
| al =   1.338e+04 (8443, 1.832e+04)||al = 1102 (-353.6, 2558)||al = 2e+04 (1.767e+04, 2.233e+04)
 
|-
 
|-
−
| mu = 7311  (6583, 8039)||mu = 1.318e+04  (1.03e+04, 1.607e+04)||mu = 4886 (3817, 5954)
+
| mu =   1.798e+04  (1.1e+04, 2.496e+04)||mu = 4252 (2395, 6109)||mu = 2.599e+04  (2.028e+04, 3.17e+04)
 
|-
 
|-
−
| n1 =      0.8499  (0.1814, 1.518)|| n1 = 0.9388 (0.5614, 1.316)||n1 = 1.09 (0.531, 1.649)
+
| n1 =      0.8499  (0.1814, 1.518)|| n1 =       1.481 (0.1392, 2.824)||n1 =     0.8996 (0.4539, 1.345)
 
|-
 
|-
 
!rowspan="5" scope="row"|Goodness to fit  
 
!rowspan="5" scope="row"|Goodness to fit  
−
|  sse: 4.2545e+07||sse: 6.4285e+08||sse: 2.7027e+08
+
|  sse: 1.1364e+10||sse: 3.7467e+08||sse: 2.0117e+10
 
|-
 
|-
−
|rsquare: 0.9964||rsquare: 0.9879||rsquare: 0.9743
+
|rsquare: 0.9586||rsquare: 0.9131||rsquare: 0.9826
 
|-
 
|-
 
|fe: 7||dfe: 7|| dfe: 7
 
|fe: 7||dfe: 7|| dfe: 7
 
|-
 
|-
−
|adjrsquare: 0.9949||adjrsquare: 0.9828||adjrsquare: 0.9633
+
|adjrsquare: 0.9408||adjrsquare: 0.8758||adjrsquare: 0.9752
 
|-
 
|-
−
|rmse: 2.4653e+03||rmse: 9.5831e+03||rmse: 6.2137e+03
+
|rmse: 4.0292e+04||rmse: 7.3160e+03||rmse: 5.3609e+04
 
|}
 
|}
  
−
<div style="margin:auto;width:840px">
+
[[File:ETH15_EngineeredPromoterinLB_low.png|thumb|center|500px|Comparison of the fluorescence levels obtained using the wild-type promoter alone and in combination with LldR and LldP-LldR]]
−
<ul>
+
−
<li class="graphthumb" style="display:inline-block;width:33%">[[Image:ETH15_P70_highLldr.png|thumb|250px|Dose-response curve for our promoter with J23118-B0034-lldR]]</li>
+
−
<li class="graphthumb" style="display:inline-block;width:33%">[[Image:ETH15_P70_lowLldPR.png|thumb|250px|Dose-response curve for our promoter with J23114-B0032-lldP-lldR]]</li>
+
−
<li class="graphthumb" style="display:inline-block;width:33%">[[Image:ETH15_P70_highLldPR.png|thumb|250px|Dose-response curve for our promoter with J23114-B0032-lldP-J23118-B0034-lldR]]</li>
+
−
</ul>
+
−
</div>
+
  
−
As you can see from the graphs, activation with only LldR is lower than with LldP-LldR. Also, the sample with LldP-LldR with a stronger promoter achieves higher fluorescence levels. We believe that this is due to the fact that more molecules of LldP are produced and therefore more lactate can enter inside the cell. Just as a qualitative example, J23118-B0034-lldR has half of the maximum fluorescence than J23118-B0034-lldP-lldR.
+
As can be seen in the picture above, when P<sub>lldR</sub> GFP is alone, the fluorescence levels do not vary with lactate concentration. However, if P<sub>lldR</sub> GFP is expressed in a system with constitutive expression of LldR, at low concentrations of lactate there is repression of the promoter, while at high concentrations there is activation. Activation is higher when there is constitutive expression of LldP-LldR.  
−
When comparing J23114-B0034-lldP-lldR and J23118-B0034-lldP-lldR, we can see that J23114-B0034-lldP-lldR has more leakiness, which we think is due to its low expression, and therefore it cannot repress the gene properly.  
+
  
−
In the figure below, you can see a comparison between the wild-type promoter and our designed promoters maintaining the same structure than the aid-type but changing the promoter for an Anderson promoter. LldR is required for the repression of the promoter in the absence of lactate, and it is also necessary for the activation in the presence of lactate.
+
The promoter has higher ON/OFF ratios than the wild-type promoter (see Table 1). Its K<sub>M</sub> is higher than the one from the wild-type promoter, meaning that is has lower affinity for the substrate.  
−
https://static.igem.org/mediawiki/parts/b/b6/ETH15_Characterization_most_promising_promoters.png
+
  
 +
This promoter is one from our designed and tested synthetic promoters based on the wild-type P<sub>lldR</sub>. In the following table, one can see the ON/OFF ratios and the K<sub>M</sub> of the wild-type promoter and the three more prominent members of the library.
 +
 +
Table 1: ON/OFF ratio
 +
{|class="wikitable"
 +
|-
 +
! !!J23118-B0034-lldR!!J23118-B0034-lldP-lldR!!J23114-B0032-lldP-lldR
 +
|-
 +
|[[Part:BBa_K822000]] (wild-type)|| 10.35||8.04||1.16
 +
|-
 +
|[[Part:BBa_K1847008]]||15.26||23.96||1.42
 +
|-
 +
|[[Part:BBa_K1847009]]||1.56||24.34||0.96
 +
|-
 +
|[[Part:BBa_K1847007]]||1.34||3.85||1.29
 +
|-
 +
|}
 +
 +
Table 2: K<sub>M</sub> (&micro;M)
 +
{|class="wikitable"
 +
|-
 +
! !!J23118-B0034-lldR!!J23118-B0034-lldP-lldR!!J23114-B0032-lldP-lldR
 +
|-
 +
|[[Part:BBa_K822000]] (wild-type)|| 955||1930||720.2
 +
|-
 +
|[[Part:BBa_K1847008]]||1075||1751||337.5
 +
|-
 +
|[[Part:BBa_K1847009]]||977.5||2361||459.8
 +
|-
 +
|[[Part:BBa_K1847007]]||697.7||1977||1337
 +
|-
 +
|}
  
 
<h1>References</h1>
 
<h1>References</h1>
Line 85: Line 107:
 
   <li>J Bacteriol. 2008 Apr; 190(8): 2997–3005.</li>
 
   <li>J Bacteriol. 2008 Apr; 190(8): 2997–3005.</li>
 
</ol>
 
</ol>
−
 
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1847008 parameters</partinfo>
 
<partinfo>BBa_K1847008 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 15:11, 17 September 2015

lldRO1-J23117-lldRO2

Collection of promoters regulated by LldR.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 78
    Illegal NheI site found at 101
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Theoretical model for the dual function as repressor and activator of LldR.

The natural promoter of LldR (Part:BBa_K822000) consists of two operators (O1 and O2) and a promoter which is intercalated between the operators. It regulates the expression of the lldPRD operon, and it is involved in L-lactate metabolism. This promoter is repressed by a dimer of LldR, possibly by forming a DNA loop that does not allow the RNA polymerase to bind to the promoter. LldR can also have a function as an activator [1]. The repression of the promoter can be removed by lactate.

Characterization of the promoter

We wanted to know how the promoter would respond to the lactate presence and how important is the architecture of the promoter in this response. For this regard, we built several promoters. Part:BBa_K1847002, Part:BBa_K1847003, Part:BBa_K1847004, Part:BBa_K1847005, Part:BBa_K1847006, Part:BBa_K1847007, Part:BBa_K1847008, and Part:BBa_K1847009.

Genetic design for characterization with LldR

To test our promoter we designed a plasmid containing the promoter with sfGFP in a medium copy plasmid pSEVA261 and transformed it into Escherichia coli TOP10. Then, we added a second plasmid containing lldR with a medium strong promoter (Part:BBa_J23118) and a strong RBS (Part:BBa_B0034) in pSEVA371 backbone. According to literature, LldR will repress the promoter and by the addition of lactate the production of GFP will start, as the repressor will be removed.

Genetic design for characterization with LldP-LldP

The second step for our characterization was to add an L-lactate permease (LldP), which despite its name is an active transporter of L-lactate, D-lactate and glycolate. To understand the effect of LldP (L-lactatep permease) in our system, we tried the following conditions:

  • Plasmid with the promoter and sfGFP in medium copy number plasmid+ J23114-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in low copy number plasmid
  • Plasmid with the promoter and sfGFP in medium copy number plasmid + J23118-B0032-lldP-lldR (LldP and LldR are under the control of the same promoter) in low copy number plasmid

Plate reader

E. coli TOP10 strains were grown overnight in Lysogeny Broth (LB) containing kanamycin (1 µ/mL) and chloramphenicol (0.36 µ/mL) at 37°C and 200 rpm. Cultures were diluted 1:50 in fresh LB with the corresponding antibiotic and transferred to a 96-well plate (200 µL/well). Cultures were grown for 90 min to arrive to exponential phase and then different concentrations of lactate were added. Samples were always made in triplicates and a blank of LB with the corresponding lactate concentration was done. During 7 h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 7 min. The plate was always kept at 37°C. We calculated dose-response curves from the exponential phase of the bacteria.

Modeling

Each experimental data set was fitted to a Hill function using the Least Absolute Residual method (Fitting Toolbox in MatLab).

ETH15 equation.png

Where:

  • al: leakiness in µM/min
  • mu: constant in µM/min
  • Km: half-maximum effective concentration (a representation of sensitivity) in µM
  • n: Hill coefficient (a representation of cooperativity), unitless


J23118-B0034-lldR J23118-B0034-lldP-lldR J23114-B0032-lldP-lldR
Coefficient values K=697.7 (75.16, 1320) K=1977 (474.5, 3479) K=1337 (470, 2203)
al = 1.338e+04 (8443, 1.832e+04) al = 1102 (-353.6, 2558) al = 2e+04 (1.767e+04, 2.233e+04)
mu = 1.798e+04 (1.1e+04, 2.496e+04) mu = 4252 (2395, 6109) mu = 2.599e+04 (2.028e+04, 3.17e+04)
n1 = 0.8499 (0.1814, 1.518) n1 = 1.481 (0.1392, 2.824) n1 = 0.8996 (0.4539, 1.345)
Goodness to fit sse: 1.1364e+10 sse: 3.7467e+08 sse: 2.0117e+10
rsquare: 0.9586 rsquare: 0.9131 rsquare: 0.9826
fe: 7 dfe: 7 dfe: 7
adjrsquare: 0.9408 adjrsquare: 0.8758 adjrsquare: 0.9752
rmse: 4.0292e+04 rmse: 7.3160e+03 rmse: 5.3609e+04
Comparison of the fluorescence levels obtained using the wild-type promoter alone and in combination with LldR and LldP-LldR

As can be seen in the picture above, when PlldR GFP is alone, the fluorescence levels do not vary with lactate concentration. However, if PlldR GFP is expressed in a system with constitutive expression of LldR, at low concentrations of lactate there is repression of the promoter, while at high concentrations there is activation. Activation is higher when there is constitutive expression of LldP-LldR.

The promoter has higher ON/OFF ratios than the wild-type promoter (see Table 1). Its KM is higher than the one from the wild-type promoter, meaning that is has lower affinity for the substrate.

This promoter is one from our designed and tested synthetic promoters based on the wild-type PlldR. In the following table, one can see the ON/OFF ratios and the KM of the wild-type promoter and the three more prominent members of the library.

Table 1: ON/OFF ratio

J23118-B0034-lldR J23118-B0034-lldP-lldR J23114-B0032-lldP-lldR
Part:BBa_K822000 (wild-type) 10.35 8.04 1.16
Part:BBa_K1847008 15.26 23.96 1.42
Part:BBa_K1847009 1.56 24.34 0.96
Part:BBa_K1847007 1.34 3.85 1.29

Table 2: KM (µM)

J23118-B0034-lldR J23118-B0034-lldP-lldR J23114-B0032-lldP-lldR
Part:BBa_K822000 (wild-type) 955 1930 720.2
Part:BBa_K1847008 1075 1751 337.5
Part:BBa_K1847009 977.5 2361 459.8
Part:BBa_K1847007 697.7 1977 1337

References

  1. J Bacteriol. 2008 Apr; 190(8): 2997–3005.