5 The role of quorum-sensing in the virulence of Pseudomonas ...
5 The role of quorum-sensing in the virulence of Pseudomonas ...
5 The role of quorum-sensing in the virulence of Pseudomonas ...
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Plasmids<br />
Plasmid Description Reference<br />
pLLX13<br />
pLLX8<br />
pWSK29<br />
lys10<br />
capture<br />
vector<br />
lys47<br />
capture<br />
vector<br />
asnV<br />
capture<br />
vector<br />
leuX<br />
capture<br />
vector<br />
serT<br />
capture<br />
vector<br />
serW<br />
capture<br />
vector<br />
(9.9kb) provides <strong>the</strong> backbone for <strong>the</strong> capture vector. It conta<strong>in</strong>s<br />
<strong>the</strong> tetAR genes that confer tetracycl<strong>in</strong>e resistance (10µg/ml) <strong>in</strong><br />
E. coli. It has <strong>the</strong> URA3 gene that will allow S. cerevisiae CRY1-<br />
2 to grow on uracil –deficient media. <strong>The</strong> plasmid also conta<strong>in</strong>s<br />
genes to allow replication <strong>in</strong> both types <strong>of</strong> organisms. CEN6 and<br />
ARSH4 allow replication and segregation <strong>of</strong> <strong>the</strong> plasmid with<strong>in</strong><br />
yeast. <strong>The</strong>re are two orig<strong>in</strong> <strong>of</strong> replication; oriV for vegetative<br />
replication <strong>in</strong> E. coli and oriT which acts as an orig<strong>in</strong> <strong>of</strong> transfer.<br />
(4.6kb) is used to produce an amplicon <strong>of</strong> 2.9kb that conta<strong>in</strong> <strong>the</strong><br />
cyh2, which confers sensitivity to cycloheximide (2.5 µg/ml) <strong>in</strong><br />
S. cerevisiae CRY 1-2 and bla (b-lactamase) confer resistance to<br />
carbenicll<strong>in</strong> (50µg/ml) <strong>in</strong> E. coli.<br />
Low copy vector used for <strong>the</strong> sub-clon<strong>in</strong>g <strong>of</strong> E106-serW genomic<br />
island<br />
Capture vector that targets <strong>the</strong> 10 th tRNA for lys<strong>in</strong>e <strong>in</strong><br />
<strong>Pseudomonas</strong> aerug<strong>in</strong>osa PAO1; adjacent to PA0976<br />
Capture vector that targets <strong>the</strong> 47 th tRNA for lys<strong>in</strong>e <strong>in</strong><br />
<strong>Pseudomonas</strong> aerug<strong>in</strong>osa PAO1; adjacent to PA4541<br />
Capture vector that targets asnV <strong>in</strong> Escherichia coli K12<br />
MG1655<br />
Capture vector that targets leuX <strong>in</strong> Escherichia coli K12<br />
MG1655<br />
35<br />
Stephen Lory<br />
Dept. <strong>of</strong> Microbiology<br />
and Molecular<br />
Genetics<br />
Harvard Medical<br />
School<br />
(Wolfgang et al 2003)<br />
Stephen Lory<br />
Dept. <strong>of</strong> Microbiology<br />
and Molecular<br />
Genetics<br />
Harvard Medical<br />
School<br />
(Wolfgang et al 2003)<br />
Lab 212<br />
Dept. <strong>of</strong> Infection,<br />
Immunity and<br />
Inflammation,<br />
University <strong>of</strong> Leicester<br />
(Wang, Kushner 1991)<br />
This study<br />
This study<br />
This study<br />
This study<br />
Capture vector that targets serT <strong>in</strong> Escherichia coli K12 MG1655 This study<br />
Capture vector that targets serW <strong>in</strong> Escherichia coli K12<br />
MG1655<br />
Table 2-6 shows plasmids used and created dur<strong>in</strong>g this study<br />
2.1.3 Preparation <strong>of</strong> electrocompetent cells<br />
This study<br />
A 5ml culture <strong>of</strong> E. coli DH10B was grown overnight 200rpm, 37ºC <strong>in</strong> LB. <strong>The</strong><br />
overnight culture was added to 495ml <strong>of</strong> LB and <strong>in</strong>cubated at 200rpm, 37ºC for<br />
approximately 2-3 hours until culture reached an OD600 <strong>of</strong> 0.5. <strong>The</strong> culture was