1. 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
JAY

2. Bacterial Conjugation
The F+ plasmid is necessary for bacterial conjugation F F F
F Pilus Formation
GABE F+ F- F+

3. 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
GABE

4. 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
GABE

5. General Problems All genes necessary, but to what degree?
traJ construct conjugates
Need to find new genes to knockout

6. 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

7. New knockouts
F plasmid
 trbC gene
R plasmid
 traG