Practicals : Aims

1
Practicals Aims

• 1. Phage restriction
• Observe phage restriction and consider the
  implications of this phenomenon in nature.
• Acquire the skills needed to cultivate and titre
  phage in practice
• 2. Phage co-transduction
• Demonstrate the close linkage of two bacterial
  genes by transductional mapping
• Observe the connection between RecA function and
  DNA repair
• 3. Conjugation and transposition
• Demonstrate plasmid conjugation
• Demonstrate Insertion Sequence transposition/coint
  egration

2
IS1 mediated excisions in the gal operon
galIS1 in this sector reverts as red WT (gal)
colonies on McConkey Agar. Note the high
frequency
gal point (amber) mutation. Supression revertants
at low frequency
?gal (deletion). No revertants possible
3
Effect of UV irradiation on the growth of E.coli
recA mutants on an agar plate
UV this side
This side covered
E.coli WT
recA mutant another recA mutant
4
Demonstration of ?CI857 phage restriction in
E.coli K12.
?CI857 titred on C600 (restriction minus) after
growth on DP1060 dcm methylase minus
?CI857 titred on C600 (restriction minus)after
growth on DP990 dcm methylase_
?CI857 titred on C600 (pN3 EcoRII restriction
plus) after growth on DP990 dcm methylase
?CI857 titred on C600 (pN3 EcoRII restriction
plus) after growth on DP1060 dcm methylase minus
5
Plasmid cointegration mediated by IS1. See
practical exercise
Crosses of A. E.coli DP990 (pOXKm, pKPG16
(pBR322IS1)) with C600 nalR B. E.coli DP990
(pOXKm, pBR322 (Control)) with C600 nalR
A. 100µl undiluted and plated on Km,Tet and Nal
agar plates. Cointegrates grow
100µl of 10-3 dilution plated on Km, Nal
plates. Indicates the pOXKm transconjugants
B. 100µl undiluted and plated on Km,Tet and Nal
agar plates. No cointegrates detected
6
Deletion of adjacent genes due to activity of IS1
inserted in the gal operon
Chlorate resistant colonies on chlorate/McConkey
agar. A. IS1 has transposed to chlD locus but
still reverts as papillae to gal B. IS1 has
transposed to chlD locus then gal has been
deleted hence no papillae
7
Deletion of adjacent genes due to activity of IS1
inserted in the gal operon
Close-up of colonies with gal papillae
Chlorate resistant colonies on chlorate/McConkey
agar. A. IS1 has transposed to chlD locus but
still reverts as papillae to gal B. IS1 has
transposed to chlD locus then gal has been
deleted hence no papillae