Team:Alberta-North-RBI E/genetics

From 2012e.igem.org

(Difference between revisions)
(Our Microscopic Friend)
Line 2: Line 2:
=='''Genetics'''==
=='''Genetics'''==
-
== Our Microscopic Friend ==
 
We plan to use a single metabolic pathway for the conversion of glucose to aromatics. We are currently engineering a Pseudomonas putida strain containing a “switch” system enabling the manufacture of either shikimic acid (SA) or cinnamic acid (CA) as a major product. Future embodiments of this strain will contain additional directing mechanisms whereby a multitude of derivative chemicals can be favored.  
We plan to use a single metabolic pathway for the conversion of glucose to aromatics. We are currently engineering a Pseudomonas putida strain containing a “switch” system enabling the manufacture of either shikimic acid (SA) or cinnamic acid (CA) as a major product. Future embodiments of this strain will contain additional directing mechanisms whereby a multitude of derivative chemicals can be favored.  
    
    
Line 24: Line 23:
<center>
<center>
<img src=" https://static.igem.org/mediawiki/2012e/9/9f/UAlberta_E_Constructs.jpg" width="767px" height="192px"; >
<img src=" https://static.igem.org/mediawiki/2012e/9/9f/UAlberta_E_Constructs.jpg" width="767px" height="192px"; >
 +
 +
 +
<TABLE WIDTH=632 BORDER=1 BORDERCOLOR="#00000a" CELLPADDING=7 CELLSPACING=0 RULES=GROUPS>
 +
<COL WIDTH=197>
 +
<COL WIDTH=65>
 +
<COL WIDTH=58>
 +
<COL WIDTH=254>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=6 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Part
 +
name</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Gene/Locus</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Source</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Note</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Constitutive
 +
promoter</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>n/a</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Synthetic</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>General
 +
part</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Inducible
 +
promoter (cis)</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>n/a</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Synthetic</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>General
 +
part</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Inducible
 +
promoter (trans)</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>n/a</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Synthetic</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>General
 +
part</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Inducible
 +
repressor (cis)</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>n/a</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Synthetic</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>General
 +
part</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Inducible
 +
repressor (trans)</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>n/a</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Synthetic</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>General
 +
part</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Ribosome
 +
binding site</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>n/a</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Synthetic</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>General
 +
part</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Transcription
 +
terminator</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>n/a</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Synthetic</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>General
 +
part</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Transketolase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>tktA</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Constitutively
 +
upregulated</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>DAHP
 +
synthase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>aroF^FBR</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Constitutively
 +
upregulated, Thr326Pro = feedback insensitive</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>DHQ
 +
synthase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>aroB</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Constitutively
 +
upregulated</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>DHQ
 +
dehydratase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>aroD</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Constitutively
 +
upregulated</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Shikimate
 +
dehydrogenase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>aroE</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Constitutively
 +
upregulated</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Shikimate
 +
kinase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>aroK</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>3-phosphoshikimate
 +
1-carboxyvinyltransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1411</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Chorismate
 +
synthase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1471</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Chorismatemutase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1410</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Aromatic
 +
amino acid aminotransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1561</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Aromatic
 +
amino acid aminotransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_3083</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Histidinol-phosphate
 +
aminotransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_0995</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=197 HEIGHT=6 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Phenylalanine
 +
ammonia-lysase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=65 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000">&nbsp;</FONT></P>
 +
</TD>
 +
<TD WIDTH=58 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>R.
 +
toruloides</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=254 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Introduced
 +
as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
</TABLE>
 +
<P STYLE="margin-bottom: 0.35cm"><BR><BR>
 +
</P>
 +
<P STYLE="margin-bottom: 0.35cm">Genes to be removed:</P>
 +
<TABLE WIDTH=632 BORDER=1 BORDERCOLOR="#00000a" CELLPADDING=7 CELLSPACING=0 RULES=GROUPS>
 +
<COL WIDTH=201>
 +
<COL WIDTH=54>
 +
<COL WIDTH=60>
 +
<COL WIDTH=260>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=6 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Shikimate
 +
kinase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>aroK</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>3-phosphoshikimate
 +
1-carboxyvinyltransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1411</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Chorismate
 +
synthase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1471</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Chorismatemutase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1410</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Aromatic
 +
amino acid aminotransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1561</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Aromatic
 +
amino acid aminotransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_3083</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Histidinol-phosphate
 +
aminotransferase</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_0995</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed
 +
and reintroduced as inducible</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
<TBODY>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Pyrroloquinolinequinone</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_3064</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Anthranilate
 +
synthase component I</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_0413</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Anthranilate
 +
synthase component II</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_0416</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Para-aminobenzoate
 +
synthase subunit I</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>PPS_1911</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=7 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Shikimate
 +
transporter</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>shiA</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2><I>P.
 +
putida</I></FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=BOTTOM>
 +
<TD WIDTH=201 HEIGHT=6 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Cinnamic
 +
acid transporter</FONT></FONT></FONT></P>
 +
</TD>
 +
<TD WIDTH=54 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000">&nbsp;</FONT></P>
 +
</TD>
 +
<TD WIDTH=60 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000">&nbsp;</FONT></P>
 +
</TD>
 +
<TD WIDTH=260 BGCOLOR="#ffffff">
 +
<P><FONT COLOR="#000000"><FONT FACE="Calibri, serif"><FONT SIZE=2>Removed</FONT></FONT></FONT></P>
 +
</TD>
 +
</TR>
 +
</TBODY>
 +
</TABLE>
 +
<P STYLE="margin-bottom: 0.35cm"><BR><BR>
</center>
</center>
</html>
</html>

Revision as of 22:52, 14 October 2012

Genetics

We plan to use a single metabolic pathway for the conversion of glucose to aromatics. We are currently engineering a Pseudomonas putida strain containing a “switch” system enabling the manufacture of either shikimic acid (SA) or cinnamic acid (CA) as a major product. Future embodiments of this strain will contain additional directing mechanisms whereby a multitude of derivative chemicals can be favored.


The enzymatic activities leading to the production of SA will be enhanced by the addition of extra-chromosomal copies of endogenous genes modified such that expression is constitutive. Where necessary, these genes will be further modified through site-directed mutagenesis to abolish feedback regulation. Optimization of metabolism to SA is based on the invention(s) described in US patents 5168056 and 6613552.


Enzymatic activities which lead from SA to phenylalanine (Phe) will be abolished by removing the underlying endogenous genes. These endogenous genes, in addition to a codon-optimized Rhodosporidium toruloides gene encoding for a phenylalanine ammonia-lysase (PAL) enzyme, will be re-introduced at an extra-chromosomal locus as modified genes containing inducible promoter/repressor “switches” allowing us to direct metabolism toward CA at will. The “switch” mechanism will require “cis” DNA elements immediately upstream of each enzyme’s coding sequence (CDS). In addition, each regulatory system (ie. induction, repression) will require a constitutively-regulated “trans” element encoded at a different locus which produces the protein component. To enhance the yield of our desired products, two additional categories of changes will be made to our host strain. Enzymatic activities which direct metabolites away from the desired pathway will be abolished by removing the underlying endogenous genes and transporter proteins responsible for re-uptake of SA or CA from the medium will be removed in the same manner.



Part name

Gene/Locus

Source

Note

Constitutive promoter

n/a

Synthetic

General part

Inducible promoter (cis)

n/a

Synthetic

General part

Inducible promoter (trans)

n/a

Synthetic

General part

Inducible repressor (cis)

n/a

Synthetic

General part

Inducible repressor (trans)

n/a

Synthetic

General part

Ribosome binding site

n/a

Synthetic

General part

Transcription terminator

n/a

Synthetic

General part

Transketolase

tktA

P. putida

Constitutively upregulated

DAHP synthase

aroF^FBR

P. putida

Constitutively upregulated, Thr326Pro = feedback insensitive

DHQ synthase

aroB

P. putida

Constitutively upregulated

DHQ dehydratase

aroD

P. putida

Constitutively upregulated

Shikimate dehydrogenase

aroE

P. putida

Constitutively upregulated

Shikimate kinase

aroK

P. putida

Removed and reintroduced as inducible

3-phosphoshikimate 1-carboxyvinyltransferase

PPS_1411

P. putida

Removed and reintroduced as inducible

Chorismate synthase

PPS_1471

P. putida

Removed and reintroduced as inducible

Chorismatemutase

PPS_1410

P. putida

Removed and reintroduced as inducible

Aromatic amino acid aminotransferase

PPS_1561

P. putida

Removed and reintroduced as inducible

Aromatic amino acid aminotransferase

PPS_3083

P. putida

Removed and reintroduced as inducible

Histidinol-phosphate aminotransferase

PPS_0995

P. putida

Removed and reintroduced as inducible

Phenylalanine ammonia-lysase

 

R. toruloides

Introduced as inducible



Genes to be removed:

Shikimate kinase

aroK

P. putida

Removed and reintroduced as inducible

3-phosphoshikimate 1-carboxyvinyltransferase

PPS_1411

P. putida

Removed and reintroduced as inducible

Chorismate synthase

PPS_1471

P. putida

Removed and reintroduced as inducible

Chorismatemutase

PPS_1410

P. putida

Removed and reintroduced as inducible

Aromatic amino acid aminotransferase

PPS_1561

P. putida

Removed and reintroduced as inducible

Aromatic amino acid aminotransferase

PPS_3083

P. putida

Removed and reintroduced as inducible

Histidinol-phosphate aminotransferase

PPS_0995

P. putida

Removed and reintroduced as inducible

Pyrroloquinolinequinone

PPS_3064

P. putida

Removed

Anthranilate synthase component I

PPS_0413

P. putida

Removed

Anthranilate synthase component II

PPS_0416

P. putida

Removed

Para-aminobenzoate synthase subunit I

PPS_1911

P. putida

Removed

Shikimate transporter

shiA

P. putida

Removed

Cinnamic acid transporter

 

 

Removed







Follow Us

Contact Us

Email: ualbertaigem@gmail.com

Search