Difference between revisions of "Part:BBa K902017"

Latest revision as of 09:34, 29 October 2012

MgtAp-mgtArb-GFP(LVA)

This part was built to characterize the control element of the magnesium killswitch with mgtAp-mgtArb. GFP is used as a reporter to indicate how well the system responds to different concentrations of magnesium compared to controls. For the protocol and experimental design please click here. For characterization data please click here. This part shows that with lower concentrations of magnesium it is activated and with higher concentrations of magnesium it is repressed.

Figure 1: The blue line indicating the response to magnesium shows a dramatic decrease at approximately 1 mM concentration of magnesium. the system completely shuts off at 1mM magnesium.

It is important to note that these constructs do not contain the signal transduction components PhoP and PhoQ and therefore the promoter riboswitch system appears to be leaky, i.e it does not completely shut off when there is 10mM magnesium.

Usage and Biology

The mgt system is used in the control and metabolism of magnesium in E. coli. It is important in regulating the transport of these ions into and out of the cell to ensure homeostasis is maintained. It has been previously characterized that the mgtA transport system contains an upstream promoter and riboswitch sensative to magnesium. We have cloned and biobricked both of these components and shown that they demonstrate the ability

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 BsaI.rc site found at 974

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 <partinfo>BBa_K902017 short</partinfo>
-<html> This part was built to characterize the control element of the magnesium killswitch with mgtAp-mgtArb. GFP is used as a reporter to indicate how well the system responds to different concentrations of magnesium compared to controls. For characterization data please click <a href="http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">here</a>. </html>[[Image:Magmesium graph.png|500px|left|Figure 3: This graph represents the relative flourescence units from the mgtA promoter riboswitch construct as well as the riboswitch construct under the TetR promoter (BBa_R0040). We can see a decrease in the level of GFP output with increasing concentrations of magnesium. There is much steeper decrease in the GFP output in the construct with the magnesium promoter and riboswitch compared to the construct with just the riboswitch alone.]]<html> <p>There is a much larger decrease in the GFP output when the mgtA promoter and riboswitch are working together compared to the mgtA riboswitch alone under the control of TetR promoter. This suggests that having both the promoter and the riboswitch together provides a tighter control over the genes expressed downstream. This also suggests that magnesium riboswitch alone is sufficient in reducing gene expression downstream of a constitutive promoter.</p>
+<html> This part was built to characterize the control element of the magnesium killswitch with mgtAp-mgtArb. GFP is used as a reporter to indicate how well the system responds to different concentrations of magnesium compared to controls. For the protocol and experimental design please click <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/mgcircuit">here</a>. For characterization data please click <a href="http://2012.igem.org/Team:Calgary/Project/HumanPractices/Killswitch/Regulation">here</a>. This part shows that with lower concentrations of magnesium it is activated and with higher concentrations of magnesium it is repressed. </html>[[Image:Magmesium graph.png|thumb|center|500px|Figure 1: The blue line indicating the response to magnesium shows a dramatic decrease at approximately 1 mM concentration of magnesium. the system completely shuts off at 1mM magnesium.]] <html> It is important to note that these constructs do not contain the signal transduction components <a href="https://parts.igem.org/BBa_K902010">PhoP</a> and <a href="https://parts.igem.org/BBa_K902011">PhoQ</a> and therefore the promoter riboswitch system appears to be leaky, i.e it does not completely shut off when there is 10mM magnesium. </html>
-<p>It is important to consider however that the control elements of the system namely PhoP and PhoQ were not present in the circuits tested and therefore there is some GFP expression in even at the inhibitory concentration (10mM MgCl2). We believe that having the regulatory elements would give us better control and get rid of the leakiness.</p>
-<p>Although the magnesium system is highly regulated, it is not a suitable system for the purposes of our bioreactor. The tailings are composed of very high concentration of magnesium- upto 120mM(Kim et al, 2011). As can be seen from figure 3, this would inhibit the system. Therefore, if our bacteria escapes into the tailings, the kill genes would not be activated and the bacteria would be able to survive.</p>
-<p>In contrast, it is important to note that this system adds important regulatory elements to the registry such as an inducible promoter and a riboswitch which can be used by other teams to control both killswitches as well as other regulatory pathways which do not pertain using tailings. </p></html>
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+The <i>mgt</i> system is used in the control and metabolism of magnesium in <i>E. coli</i>.  It is important in regulating the transport of these ions into and out of the cell to ensure homeostasis is maintained.  It has been previously characterized that the <i>mgtA</i> transport system contains an upstream promoter and riboswitch sensative to magnesium.  We have cloned and biobricked both of these components and shown that they demonstrate the ability
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