Difference between revisions of "Part:BBa K540001"

m
 
(18 intermediate revisions by 4 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K540001 short</partinfo>
 
<partinfo>BBa_K540001 short</partinfo>
  
−
Prcn is a promoter that is activated by cobalt. The RcnR repressor binds to the promoter by default. When enough cobalt molecules accumulate in the cell cytoplasm, the cobalt ions bind to the RcnR repressor and prevent its fixation on the rcn promoter.
+
P<i>rcn</i> is a promoter that is activated by cobalt. The RcnR repressor binds to the promoter by default. When enough cobalt molecules accumulate in the cell cytoplasm, the cobalt ions bind to the RcnR repressor and prevent its fixation on the rcn promoter.
  
 
Prcn is also activated by nickel, but we have not characterized this aspect.
 
Prcn is also activated by nickel, but we have not characterized this aspect.
Line 14: Line 14:
  
  
−
<b>Important : this characterization hasn't been done with this biobrick and an iGEM Gfp. It has been done with the  wild type promoter. The part we provide contain the exact sequence of the promoter but a silent mutation to remove a PstI site. The GFP used here, is a stable GFP.</b>  
+
<b>Important : this characterization hasn't been done with this biobrick and an iGEM Gfp. It has been done with the  wild type promoter. The part we provide contains the exact sequence of the promoter but a silent mutation to remove a PstI site. The GFP used here, is a stable GFP.</b>  
  
 
In order to determine wether <i>rcn</i> promoter is inducible by cobalt or not, a kinetic of the fluorescence (excitation 490nm, emission 510nm) of the two following strains has been done:<br/>
 
In order to determine wether <i>rcn</i> promoter is inducible by cobalt or not, a kinetic of the fluorescence (excitation 490nm, emission 510nm) of the two following strains has been done:<br/>
−
NM522/p157 where p157 is a plasmid containing a stable GFP under the control of <i>rcn</i> promoter.<br/>
+
NM522/p157 where p157 is a plasmid containing a fusion P<i>rcn</i>-stable GFP (= pPROBE OT′ carrying the gfp coding sequence under the control of the rcnA promoter, described in Appl Environ Microbiol. 2009 March; 75(6): 1723–1733). <br/>
−
NM522/p115 where p115 is the p157 corresponding control plasmid.
+
NM522/p115 where P115 is the control plasmid pPROBE OT′.
  
−
Both strain are cultivated with (25µM) or without cobalt.
+
Both strains are cultivated with (25µM) or without cobalt.
 
1ml of bacteria is added to a mix of 100ml of M63G medium + 1ml of spectinomycin in a 250ml erlen.
 
1ml of bacteria is added to a mix of 100ml of M63G medium + 1ml of spectinomycin in a 250ml erlen.
 
The erlens are put in a 37°C bath with a 270rpm agitation.
 
The erlens are put in a 37°C bath with a 270rpm agitation.
−
Every hour, a 3mL and 1mL culture sampling are done for a fluorescence measurement and a OD(600nm) measurement respectively. Near an OD(600nm) of 1, samplings are done every half hour.
+
Every hour, a 3mL and 1mL culture sampling are done for a fluorescence measurement and an OD(600nm) measurement respectively. As we expected a peak of expression during the early exponential phase (personnal communication of Camille Bleriot),samplings are done every half hour during that time.
 
The measurements are stopped when the OD reaches 1. A last sampling is done after an overnight growth.
 
The measurements are stopped when the OD reaches 1. A last sampling is done after an overnight growth.
  
−
The experiment has been performed in triplicate and the results shown below are the mean values obtained from the 3 operators.
+
The experiment has been performed in triplicate by 3 different students during the same day. The results shown below are the mean values obtained from the 3 operators.
  
 
<br/>
 
<br/>
−
https://static.igem.org/mediawiki/2011/c/c8/INSA-ENS_RCN-GFP_FluoDo%3Dft.png
+
https://static.igem.org/mediawiki/parts/7/7f/INSA-ENS_Prcn-Gfp_FluoDo%3Dft.png
−
<br/>
+
−
https://static.igem.org/mediawiki/2011/3/30/INSA-ENS_RCN-GFP_FluoDo%3DfDo.png
+
  
−
It is shown out from these two graphics that the strain NM522/p115 (carrying a control plasmid) doesn't have any fluorescent activity and does not respond at all to cobalt. Whereas the strain NM522/p157 (carrying a plasmid with GFP under the control of <i>rcn</i> promoter) responds well to cobalt. A fluorescent activity is detected even without cobalt. This could be linked to a leaky nature of the promoter rcn. However this activity is at least two times lower than the one obtained in presence of cobalt.
 
  
  
 +
This graph shows that the strain NM522/p115 (carrying a control plasmid) exhibits only residual fluorescent activity and does not respond at all to cobalt. Whereas the strain NM522/p157 (carrying a plasmid with GFP under the control of <i>rcn</i> promoter) responds spectacularly well to cobalt: a drastic increase of the fluorescent specific activity is observed in presence of the metal.  Moreover, we observed that a fluorescent activity of the Prcn-gfp can be detected even without added cobalt (induction factor of 3 rsulting from cobalt addition). This probably results from the leaky nature of the promoter <i>rcn</i>(Camille Bleriot, personal communication).
  
−
<b>Conclusion</b>
+
 
 +
 
 +
<FONT COLOR="RED"><b>Conclusion :</b></FONT>
  
 
Our characterisation of <i>rcn</i> promoter shows that this promoter is inducible by cobalt. Kinetic analysis of the promoter allowed us to establish that an OD(600nm) of 0,2 was optimum for <i>rcn</i> promoter activation.  
 
Our characterisation of <i>rcn</i> promoter shows that this promoter is inducible by cobalt. Kinetic analysis of the promoter allowed us to establish that an OD(600nm) of 0,2 was optimum for <i>rcn</i> promoter activation.  
Line 46: Line 46:
 
<br/>
 
<br/>
 
<b>NB</b> : Corroboring results of this characterisation can be seen on the main page of an other part characterisation, the part  
 
<b>NB</b> : Corroboring results of this characterisation can be seen on the main page of an other part characterisation, the part  
−
BBa_K540000,<a  href="https://parts.igem.org/Part:BBa_K540000"/>here</a>.  
+
BBa_K540000,<a  href="https://parts.igem.org/Part:BBa_K540000#rcn_adherence"/>here</a>.
 +
</html>
 +
 
 +
<html>
 +
<br/>
 +
<b>Further experiment : </b> In order to check that the cobalt's activation is selective to the rcn promoter, an other kinetic mesurment is done with a MC4100 strain transformed by a plasmid containing the pLac promoter fused with a GFP.<br/>
 +
Culture condition : 100ml of M63G medium + 1ml of an overnight culture + 1ml ampicilline in a 250ml erlen. 37°C and 250rpm.<br/>
 +
Every hour, a 3mL and 1mL culture sampling are done for a fluorescence measurement and an OD(600nm) measurement respectively.<br/><br/>
 +
<IMG SRC="https://static.igem.org/mediawiki/parts/4/49/PLac-GFP_kinetic.png" ; ALT="pLac-GFP" ; width=50%; />
 +
<br/>
 +
<br/>
 +
These results show that cobalt hasn't activated the pLac promoter. So, the cobalt activation is specific to the rcn promoter.
 
</html>
 
</html>
  
Line 61: Line 72:
  
  
−
<!-- Uncomment this to enable Functional Parameter display
+
 
−
===Functional Parameters===
+
 
 +
==Functional Parameters: Austin_UTexas==
 +
<html>
 +
<body>
 
<partinfo>BBa_K540001 parameters</partinfo>
 
<partinfo>BBa_K540001 parameters</partinfo>
−
<!-- -->
+
<h3><center>Burden Imposed by this Part:</center></h3>
 +
<figure>
 +
<div class = "center">
 +
<center><img src = "https://static.igem.org/mediawiki/parts/f/fa/T--Austin_Utexas--no_burden_icon.png" style = "width:160px;height:120px"></center>
 +
</div>
 +
<figcaption><center><b>Burden Value: 0.2 ± 5.4% </b></center></figcaption>
 +
</figure>
 +
<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
 +
<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>
 +
<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
 +
</body>
 +
</html>

Latest revision as of 03:39, 26 August 2020

rcn, cobalt-sensitive promoter

Prcn is a promoter that is activated by cobalt. The RcnR repressor binds to the promoter by default. When enough cobalt molecules accumulate in the cell cytoplasm, the cobalt ions bind to the RcnR repressor and prevent its fixation on the rcn promoter.

Prcn is also activated by nickel, but we have not characterized this aspect.

Characterization


Kinetic Mesurement


Important : this characterization hasn't been done with this biobrick and an iGEM Gfp. It has been done with the wild type promoter. The part we provide contains the exact sequence of the promoter but a silent mutation to remove a PstI site. The GFP used here, is a stable GFP.

In order to determine wether rcn promoter is inducible by cobalt or not, a kinetic of the fluorescence (excitation 490nm, emission 510nm) of the two following strains has been done:
NM522/p157 where p157 is a plasmid containing a fusion Prcn-stable GFP (= pPROBE OT′ carrying the gfp coding sequence under the control of the rcnA promoter, described in Appl Environ Microbiol. 2009 March; 75(6): 1723–1733).
NM522/p115 where P115 is the control plasmid pPROBE OT′.

Both strains are cultivated with (25µM) or without cobalt. 1ml of bacteria is added to a mix of 100ml of M63G medium + 1ml of spectinomycin in a 250ml erlen. The erlens are put in a 37°C bath with a 270rpm agitation. Every hour, a 3mL and 1mL culture sampling are done for a fluorescence measurement and an OD(600nm) measurement respectively. As we expected a peak of expression during the early exponential phase (personnal communication of Camille Bleriot),samplings are done every half hour during that time. The measurements are stopped when the OD reaches 1. A last sampling is done after an overnight growth.

The experiment has been performed in triplicate by 3 different students during the same day. The results shown below are the mean values obtained from the 3 operators.


INSA-ENS_Prcn-Gfp_FluoDo%3Dft.png


This graph shows that the strain NM522/p115 (carrying a control plasmid) exhibits only residual fluorescent activity and does not respond at all to cobalt. Whereas the strain NM522/p157 (carrying a plasmid with GFP under the control of rcn promoter) responds spectacularly well to cobalt: a drastic increase of the fluorescent specific activity is observed in presence of the metal. Moreover, we observed that a fluorescent activity of the Prcn-gfp can be detected even without added cobalt (induction factor of 3 rsulting from cobalt addition). This probably results from the leaky nature of the promoter rcn(Camille Bleriot, personal communication).


Conclusion :

Our characterisation of rcn promoter shows that this promoter is inducible by cobalt. Kinetic analysis of the promoter allowed us to establish that an OD(600nm) of 0,2 was optimum for rcn promoter activation.

To conclude, this work shows that this promoter can be used with efficiency in a cobalt sensitive system.


NB : Corroboring results of this characterisation can be seen on the main page of an other part characterisation, the part BBa_K540000,here.


Further experiment : In order to check that the cobalt's activation is selective to the rcn promoter, an other kinetic mesurment is done with a MC4100 strain transformed by a plasmid containing the pLac promoter fused with a GFP.
Culture condition : 100ml of M63G medium + 1ml of an overnight culture + 1ml ampicilline in a 250ml erlen. 37°C and 250rpm.
Every hour, a 3mL and 1mL culture sampling are done for a fluorescence measurement and an OD(600nm) measurement respectively.

pLac-GFP

These results show that cobalt hasn't activated the pLac promoter. So, the cobalt activation is specific to the rcn promoter.

Safety

This part is not toxic by itself. However, when using this part, you will probably need to handle cobalt. Cobalt is toxic in all its forms ( ionic or metallic ) by inhalation, ingestion or contact. Wear adapted personal protection equipment ( labcoat, safety glasses, safety gloves ) and dispose of it in appropriate waste containers.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 47
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Functional Parameters: Austin_UTexas

BBa_K540001 parameters

Burden Imposed by this Part:

Burden Value: 0.2 ± 5.4%

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.