Once upon a time strain 1 induced a hypersensitive reaction in Lycopersicon pinnellii' Strain 2 was

1
Once upon a time strain 1 induced a
hypersensitive reaction in Lycopersicon
pinnellii. Strain 2 was not so lucky and
molecular biologists looked at it as very boring.
However, the molecular biologist decided to try
something that made strain 2 very happy. They
tried to take genes from strain 1 and put them
into strain 2 and see if they could induce an HR.
2
So the molecular biologists purified DNA from
strain 1 and did an incomplete digestion with a
frequent cutting enzyme such as SauIIIA to obtain
random pieces, put the DNA into cosmids and
infected E. coli or put it into or vectors
(plasmids) and transformed E. coli.
avrX
Strain 1 DNA
SauIIIA
Insert with avrX
3
Once the molecular biologists had these clones
they then mated them into strain 2 and tested for
a phenotype (What kind of fairy tale uses this
word?) as seen in the next slide
Strain 2
E. coli with insert from strain 1
HB101
pRK2013 Helper plasmid
PolA dependent
Triparental mating Select on Tc,Km,Nal medium
Strain 2
4
The scientists and transconjugant strain 2 were
very happy when they determined that
conferred the ability for strain 2 to cause an
HR. Now strain 2 was almost as happy as strain 1
(not quite), but the molecular biologist was
happiest of all. How about you?
Strain 2
Strain 2 transconjugant
C
5
Transposon mutagenesis
HB101
Target plasmid
Contains transposase gene
Km
pHoKm Gus
pShe
PolA independent
Transform
PolA dependent
C2110
HB101
HB101
PolA- NalR
Km
pRK2013 Helper plasmid
pHoKm Gus
pShe
PolA dependent
C2110
Km
Triparental mating Select on Tc,Km,Nal medium