Implementation

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
2
Bacterial Conjugation

• The F plasmid is necessary for bacterial
  conjugation

F Pilus Formation
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

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

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