Team:UC Davis/Parts

From 2009.igem.org

(Difference between revisions)
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</span></b><span
</span></b><span
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">An
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">An
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exogenous gene (Ice Nucleation Protein)
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exogenous gene,
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found to be expressed on the surface of E.Coli.&nbsp; (The NC stands
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Ice-nucleation protein (INP) from <span style="font-style: italic;">Pseudomonas
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for the
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syringae</span> was suggested to
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shortened version of the entire gene, which works perfectly in E.Coli).
+
-
Ice-nucleation protein (INP) from Pseudomonas Syringae was suggested to
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be used
be used
for display of foreign proteins on the surface of <i>E. coli</i>(7).
for display of foreign proteins on the surface of <i>E. coli</i>(7).
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Furthermore, researches have shown that an INP derivative constituting
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Furthermore, studies have shown that an INP derivative constituting
the
the
N-and C-terminal domains can and has been used to display foreign
N-and C-terminal domains can and has been used to display foreign
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harness
harness
and make use of this feature by fusing a specific protein to it. <br>
and make use of this feature by fusing a specific protein to it. <br>
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<i>We have modified this protein to Biobrick standard, Tom Knights
+
<i>We have modified this protein to be consistent with BBF RFC-12
Standard.<br>
Standard.<br>
For more information go to: <a
For more information go to: <a
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style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">OmpA
is one of the proteins on the outer
is one of the proteins on the outer
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membrane of <i>E. coli</i> (13). OmpA has been found to be useful as
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membrane of <i>E. coli</i> (13),it is used as a displaying fusion
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utilizable
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protein on the cell surface . This part has already been documented on
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fusion part that can fuse our protein to and display on the surface of <i>E.
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the parts
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coli</i>.and readily available as a biobrick part to test it's efficacy
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registry; however, it has not been tested as a compnent of secretion
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versus
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system (via fusion with a target
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INP in fusion to SS. This part has already been documented on the parts
+
-
registry; however, it has not been tested via fusion with a target
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protein
protein
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linked with a cleavable signal sequence. <o:p></o:p></span><br>
+
linked with a cleavable signal sequence) <o:p></o:p></span><br>
<i><span
<i><span
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;">We
+
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"></span></i><span
-
have
+
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i>We
-
modified this protein to Biobrick standard, Tom Knights Standard.</span></i><span
+
have modified this protein to be consistent with BBF RFC-12 Standard.</i></span><br>
-
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> <br>
+
<span style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i>Note:
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<i>Note: “It has remained essentially unknown how proteins of E. coli
+
“It has remained essentially unknown how proteins of E. coli
outer
outer
membrane are sorted and incorporated into this membrane” (10)</i> <br>
membrane are sorted and incorporated into this membrane” (10)</i> <br>
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Fluorescent Protein)</span></b><span
Fluorescent Protein)</span></b><span
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> :
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"> :
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Superfolder, which will let this protein
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Mutant of GFP known to be very stable (superfolder), which will let
 +
this protein
fold quickly so we can use either a fluorescent reader or UV light to
fold quickly so we can use either a fluorescent reader or UV light to
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detect.
+
detect it. Therefore it has been used as a reporter in our secretion
 +
system.
It also serves as a small protein in testing our secretion system.<br>
It also serves as a small protein in testing our secretion system.<br>
<i>For more informaiton go to: </i><a
<i>For more informaiton go to: </i><a
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fusion
fusion
protein to ‘secrete’ from INPNC. We will do the same with OmpA. <br>
protein to ‘secrete’ from INPNC. We will do the same with OmpA. <br>
-
<i>We have modified this protein to Biobrick standard, Tom Knights
+
</span><span
-
Standard.<br>
+
style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i>We
-
For more infromation go to :<a
+
have modified this protein to be consistent with BBF RFC-12 Standard.</i></span><br>
 +
<span style="font-size: 12pt; font-family: &quot;Times New Roman&quot;,&quot;serif&quot;;"><i>For
 +
more infromation go to :<a
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"><span
href="http://partsregistry.org/wiki/index.php/Part:BBa_K265002"><span
style="color: blue;">BBa_K265002</span></a></i> <o:p></o:p></span></p>
style="color: blue;">BBa_K265002</span></a></i> <o:p></o:p></span></p>

Revision as of 00:36, 15 October 2009

 
Parts:  
Parts related to secretion:                                                                                                                            Parts related to pH sensor:
Proteins:
Promoters:
Others:
Proteins:
      Promoters:
 

INPNC: An exogenous gene, Ice-nucleation protein (INP) from Pseudomonas syringae was suggested to be used for display of foreign proteins on the surface of E. coli(7). Furthermore, studies have shown that an INP derivative constituting the N-and C-terminal domains can and has been used to display foreign proteins on the surface of E. coli(9). In our project we are intending to harness and make use of this feature by fusing a specific protein to it.
We have modified this protein to be consistent with BBF RFC-12 Standard.
For more information go to: BBa_K265008 


OmpA: OmpA is one of the proteins on the outer membrane of E. coli (13),it is used as a displaying fusion protein on the cell surface . This part has already been documented on the parts registry; however, it has not been tested as a compnent of secretion system (via fusion with a target protein linked with a cleavable signal sequence)
We have modified this protein to be consistent with BBF RFC-12 Standard.
Note: “It has remained essentially unknown how proteins of E. coli outer membrane are sorted and incorporated into this membrane” (10)
For more information go to: BBa_K103006


RBSRibosome Binding site number 32 (BBa_J61132) from the registry is being used in our secretion system.
For more information go to:
BBa_J61132


Terminator: We are using BBa_B0015, a double terminator, as our terminator in both our secretion and pH system.
For more information go to: BBa_B0015


GFP (Green Fluorescent Protein) : Mutant of GFP known to be very stable (superfolder), which will let this protein fold quickly so we can use either a fluorescent reader or UV light to detect it. Therefore it has been used as a reporter in our secretion system. It also serves as a small protein in testing our secretion system.
For more informaiton go to: BBa_K265003 


Luciferase: Luciferase is a firefly protein that also fluoresces, so it serves as a reporter as well as a testable large protein.
For more information go to: BBa_1712019


LacI: One inducible Promoter which was found in the part registry.
For more information go to: BBa_R0010


SS:It allows protein fused to outer membrane proteins to become cleaved free. In this experiment this signal sequence when placed between INPNC, contains a cleavable site that allows the target fusion protein to ‘secrete’ from INPNC. We will do the same with OmpA.
We have modified this protein to be consistent with BBF RFC-12 Standard.
For more infromation go to :BBa_K265002


6-His Tag:The 6-Histidine Tag serves as a tag for Western Blotting if our fluorescent reporters are not expressed as highly as we would like.
Note: We are using this tag, just in case if the GFP or Luciferase does not work under a plate reader.


ChvI promoter: Gene fusion studies confirmed that ChvI gene was induced by acidic conditions (1). Also, it has been known to implicate in virulence (1). This gene is one of the candidates to be use in our biological pH sensor as a promoter.


KatA promoter :This Chromosomal gene is located on the linear chromosome (2) and it seems to be induced under an acidic environment as well as being involved in the Agrobacterium tumorigenesis (2).Research has suggested that ChvG is needed for "responsiveness of  gene expression to low pH "(2). This gene has become a candidate to complete our pH sensor device from this evidence.


AopB promoter: This Chromosomal gene located on the circular chromosome (2) encodes an outer member protein exposed on the bacterial cell surface (2). Also, ChvG was shown to be absolutely required for this gene expression (2)It seems to get induced under an acidic environment as well as being involved in the Agrobacterium tumorigenesis (2). Therefore, we have chosen this gene to be one of our candidates to complete our pH sensor device.


PhoA promoter: There has been a suggestion that ChvI can activate AP activity by activating transcription of this gene, PhoA (3). Therefore, this gene has become one of our candidates to complete our pH sensor device.


ImpA promoter:Gene fusion studies confirmed that impA genes was induced by acidic conditions (1), therefore, this is one of our candidates to complete our pH sensor device.