Difference between revisions of "Part:BBa K2586007"
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2586007 short</partinfo>
 
<partinfo>BBa_K2586007 short</partinfo>
 +
<html>
 +
<p align="justify">
  
 
This part encodes for the 3-phosphoshikimate 1-carboxyvinyltransferase.  
 
This part encodes for the 3-phosphoshikimate 1-carboxyvinyltransferase.  
−
The encoded enzyme is needed for the biosynthesis of essential aromatic amino acids in the shikimate pathway. Three essential aromatic amino acids are products of the pathway: Trp, Phe and Tyr. <i>aroA</i> is expressed in the <i> E.coli</i> wildtype. <br/>  
+
The encoded enzyme is needed for the biosynthesis of essential aromatic amino acids in the shikimate pathway. Three essential aromatic amino acids are products of the pathway: Trp, Phe and Tyr. <i>aroA</i> is expressed in the <i> E.coli</i> wildtype. <br> <br>
 
<i>aroA</i> encodes the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which is the main target of the herbicide glyphosate and is strongly inhibited through glyphosate application. An amplification of <i>aroE/aroA</i> allows for glyphosate resistance (1), this is used in multiple GM plants to increase the yield and improve farming applications.
 
<i>aroA</i> encodes the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which is the main target of the herbicide glyphosate and is strongly inhibited through glyphosate application. An amplification of <i>aroE/aroA</i> allows for glyphosate resistance (1), this is used in multiple GM plants to increase the yield and improve farming applications.
 
If exposed to glyphosate, <i>E.coli</i> mutants will make changes in the original sequence of <i>aroA</i> to avoid the lethal effects of the substance.  
 
If exposed to glyphosate, <i>E.coli</i> mutants will make changes in the original sequence of <i>aroA</i> to avoid the lethal effects of the substance.  
 +
</p><br>
 +
 +
 +
<img src="https://static.igem.org/mediawiki/parts/1/17/T--Goettingen--BBa_K2586007_aroAstructure.png" alt="http://www.rcsb.org/structure/1VR6" style="float:right; width: 25%;">
 +
 +
 
<style type="text/css">
 
<style type="text/css">
 
.tg  {border-collapse:collapse;border-spacing:0;}
 
.tg  {border-collapse:collapse;border-spacing:0;}
Line 52: Line 59:
 
   </tr>
 
   </tr>
 
</table>
 
</table>
 +
</html>
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 59: Line 68:
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2586007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2586007 SequenceAndFeatures</partinfo>
−
 
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
Line 65: Line 73:
 
<partinfo>BBa_K2586007 parameters</partinfo>
 
<partinfo>BBa_K2586007 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
 +
==Characterization==
 +
===<i>aroE</i> is essential in <i>B. subtilis </i>===
 +
<html>
 +
<p align="justify">
 +
 +
As essential bacterial genes appear to be more conserved than nonessential genes, the aroE gene probably does not permit the accumulation of mutations, which can be beneficial (Jordan et al., 2002). To confirm that the aroE gene is indeed essential, we transformed the B. subtilis with a PCR product consisting of DNA fragments flanking the target gene and the intervening spc spectinomycin gene (aroE::spc deletion cassette) and propagated the bacteria on SP rich medium plates (see Notebook). Several tiny colonies appeared on the transformation plates after 24 h of incubation. However, the potential transformants were not viable and supplementation of the agar plates with casamino acids also did not improve growth of the bacteria. Thus, under the tested conditions aroE seems to be essential in B. subtilis. Next, we tested whether the chromosomal copy of the aroE gene becomes dispensable for the bacteria carrying an EPSP synthase gene on a plasmid. For this purpose, we transformed the B. subtilis wild type strain 168 with the plasmids pIGEM2 and pBP143 containing the aroE and aroA EPSP synthase genes from B. subtilis and E. coli, respectively. The wild type carrying the empty plasmid pBQ200 served as a control. Next, we transformed the three strains with the aroE::spc deletion cassette and with chromosomal DNA of strain BP233 (gltT::spc), of which the latter served as the positive control. While we did not get transformants without DNA, many transformants appeared with chromosomal DNA of strain BP233 (Figure X). Moreover, the chromosomal copy of the aroE gene was only dispensable in strains carrying an extra EPSP synthase gene on a plasmid. To conclude, the aroE EPSP synthase gene is essential in B. subtilis. Moreover, the bacteria seem to require the enzymatic function of the EPSP synthase because also the enzyme from E. coli permits growth of the aroE mutant
 +
</p>
 +
<br style="clear: both" />
 +
</html>

Revision as of 16:10, 23 September 2018

aroA: 3-phosphoshikimate 1-carboxyvinyltransferase

This part encodes for the 3-phosphoshikimate 1-carboxyvinyltransferase. The encoded enzyme is needed for the biosynthesis of essential aromatic amino acids in the shikimate pathway. Three essential aromatic amino acids are products of the pathway: Trp, Phe and Tyr. aroA is expressed in the E.coli wildtype.

aroA encodes the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, which is the main target of the herbicide glyphosate and is strongly inhibited through glyphosate application. An amplification of aroE/aroA allows for glyphosate resistance (1), this is used in multiple GM plants to increase the yield and improve farming applications. If exposed to glyphosate, E.coli mutants will make changes in the original sequence of aroA to avoid the lethal effects of the substance.


http://www.rcsb.org/structure/1VR6
Locus BSU29750
Isoelectric point 5.34
Molecular weight 39.38 kDa
Protein length 358 aa
Gene length 1074 bp
Function biosynthesis of aromatic amino acids
Product 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase/
chorismate mutase-isozyme 3
Essential no
Synonyms aroG


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 346
    Illegal AgeI site found at 787
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1021


Characterization

aroE is essential in B. subtilis

As essential bacterial genes appear to be more conserved than nonessential genes, the aroE gene probably does not permit the accumulation of mutations, which can be beneficial (Jordan et al., 2002). To confirm that the aroE gene is indeed essential, we transformed the B. subtilis with a PCR product consisting of DNA fragments flanking the target gene and the intervening spc spectinomycin gene (aroE::spc deletion cassette) and propagated the bacteria on SP rich medium plates (see Notebook). Several tiny colonies appeared on the transformation plates after 24 h of incubation. However, the potential transformants were not viable and supplementation of the agar plates with casamino acids also did not improve growth of the bacteria. Thus, under the tested conditions aroE seems to be essential in B. subtilis. Next, we tested whether the chromosomal copy of the aroE gene becomes dispensable for the bacteria carrying an EPSP synthase gene on a plasmid. For this purpose, we transformed the B. subtilis wild type strain 168 with the plasmids pIGEM2 and pBP143 containing the aroE and aroA EPSP synthase genes from B. subtilis and E. coli, respectively. The wild type carrying the empty plasmid pBQ200 served as a control. Next, we transformed the three strains with the aroE::spc deletion cassette and with chromosomal DNA of strain BP233 (gltT::spc), of which the latter served as the positive control. While we did not get transformants without DNA, many transformants appeared with chromosomal DNA of strain BP233 (Figure X). Moreover, the chromosomal copy of the aroE gene was only dispensable in strains carrying an extra EPSP synthase gene on a plasmid. To conclude, the aroE EPSP synthase gene is essential in B. subtilis. Moreover, the bacteria seem to require the enzymatic function of the EPSP synthase because also the enzyme from E. coli permits growth of the aroE mutant