Biology Reference
In-Depth Information
Plasmid preparation
Promoter DNA preparation
Terminator
pGFPAmy
Promoter region
(minimum 400 bp)
pBaSysbioll
RBS
Upstream gene
Gene of interest
PCR amplification
my
Sma
1
GFP start codon
RBS
LIC tail
LIC tail
ACTTTT
ACCGCGGGCTTTCCCGGGAAGGAGGAACT
ACT
ATGCGTAAA
TGAAAA
TGGCGCCCGAAAGGGCCCTTCCTCCTTGA
TGA
TACGCATTT
CCGCGGGCTTTCCCAGC
GGCGCCCGAAAGGGTCG
GGTGGGAAGGAGGAAC
CCACCCTTCCTCCTTG
Promoter region
Sma
1 restriction
T4 DNA polymerase + dTTP
ACTTTT
ACCGCGGGCTTTCCC GGGAAGGAGGAACT
ACT
ATGCGTAAA
TGAAAA
TGGCGCCCGAAAGGG CCCTTCCTCCTTGA
TGA
TACGCATTT
CCGCGGGCTTTCCCAGC
TCG
GGT
CCACCCTTCCTCCTTG
T4 DNA polymerase + dATP
Promoter region
ACTTTT
A GGGAAGGAGGAACT
ACT
ATGCGTAAA
TGAAAA
TGGCGCCCGAAAGGG A
TGA
TACGCATTT
Mix prepared plasmid and insert
Anneal at room temperature and transform into
E.coli
ACTTTT
A
CCGCGGGCTTTCCCAGC
TGAAAA
TGGCGCCCGAAAGGG
TCG
GGT
GGGAAGGAGGAACT
ACT
ATGCGTAAA
CCACCCTTCCTCCTTG
A
TGA
TACGCATTT
Promoter region
FIGURE 1.1
Promoter fragment and plasmid preparation for ligation-independent cloning. Plasmids
pBaSysBioII and pGFPAmy both encode the gfpmut3 reporter gene (GFP), a ribosome
binding site (RBS) optimized for use in B. subtilis and a ligation-independent cloning (LIC) site
located upstream of the RBS. Cleavage of either plasmid with SmaI followed by treatment with
T4 DNA polymerase in the presence of dATP yields a linear plasmid with non-cohesive single-
stranded ends. Promoter-containing fragments are generated by PCR using oligonucleotides
(orange arrow) with a LIC tail (bent blue tail). Treatment of the promoter-containing fragment
with T4 polymerase in the presence of dTTP yields linear fragments with non-cohesive
single-stranded ends. The single-stranded regions of the linearized plasmid and promoter-
containing DNA fragment are complementary so that upon annealing, a circular plasmid
molecule is generated with directional insertion of the promoter so that it reads into the
reporter gfp gene. This annealed plasmid transforms E. coli at high frequency despite being
nicked. Moreover, no false positive clones are obtained, as the single-strand ends of the
plasmid are non-cohesive.
reporter genes were cloned into plasmids that direct chromosomal integration by a
double-crossover event at the
amyE
(plasmids pGFPamy, pCFPamy, pYFPamy) or
the
bglS
(pGFPbglS, pCFPbglS, pYFPbglS) loci (
Bisicchia
et al.
, 2010
). Each
reporter gene has a ribosome binding site optimized for translation in
B. subtilis
,
upstream of which is located a LIC cloning site (
Figure 1.1
). Integrants with the
desired promoter fusions located at the
amyE
or
bglS
loci can be screened by a plate