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Implementation
Goal: To transfer genetic information
from one bacterial cell to another
Means: Bacterial Conjugation
Goal: To specifically control who can
read the message
Means: Riboregulation
Bacterial Conjugation
• The F+ plasmid is necessary for bacterial conjugation
F
F+
FF Pilus Formation
F
F+-
Why we used F and RP4 Plasmids
•F and RP4 exhibit differing pili lengths, biasing the order in which F and RP4
will conjugate
•F and RP4 do no conjugate with themselves
•F and RP4 are among the most studied and well-characterized conjugative
plasmids
•F and RP4 plasmids are readily available
Conjugative Plasmid Facts
• Conjugative plasmids are very large, from 60k – 100k basepairs
long
•The Origin of Transfer (OriT) is where all DNA transfer begins during
conjugation
•Knocking out, by recombination, TraJ on the F plasmid, proved
ineffective in stopping conjugation
General Problems
 All genes necessary, but to what degree?
 traJ construct conjugates
 Need to find new genes to knockout
New Knockouts:
What are we looking for?
 Gene essential to conjugation that:
 Will decrease conjugation by 7 orders of
magnitude
 Can be complemented in trans to restore
conjugation
New knockouts
F plasmid
 trbC gene
R plasmid
 traG