nti (15.11.16.)
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The entire panel of the Vector NTI software
Visualized map of a selected
vectorGeneral information
window for the selected
vector
The entire nucleotide
sequence of the selected
vector
We use the Vector NTI software for bioinformatic works such as designing and editing vectors & primers, planning strategies for plasmid
digestions etc.
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I enlarged the map of the pcGlobin2-SB100
plasmid. It is the plasmid that we used for site-
directed mutagenesis to introduce designed
mutations in the coding sequence of the SB100
transposase.
Features of the plasmid:
CMV promoter: it drives the expression of theSB100 transposase
SB100 CDS: the coding sequence of the SB100
transposase
pA (polyA – polyadenylation site): a termination
signal of the transcription of SB100 transposase
gene
These elements create an unit we call
transcriptional unit. This unit provides theexpression of the SB100 transposase gene.
Elements for the maintenance of the plasmid in
bacterial cells
Ori: this is the replication origin of the plasmid, the
site where the replication of the plasmid begins in
the bacteria.
Amp CDS: the ampicillin resistance gene for the
selection of plasmid bearing bacterial cells.
The creator of the plasmid did not assign the
promoter of the resistance gene.
---
SV40 promoter + Neo CDS + SV40 pA:
It is an another transcriptional unit that we will not
exploit during the experiment.
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Now I would like to show you where a
designed mutation (H178F) positioned in
the CDS of the SB100 transposase gene.
I assigned the sequence of the SB100
transposase gene shown in green.
Go to the next page…
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Here is the window (Ctrl + F)
for the quick search of any
given sequence located in the
plasmid.
I inserted the forward primer
sequence of the H178F site-
directed mutagenesis project
shown in red.
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Here you can see the affected
nucleotide triplet (CAC) in the
SDM shown in blue.
CAC is the nucleotide triplet
for histidine. We modified
(mutated) this triplet to TTC
to encode phenylalanine
instead of histidine.
The primer contains TTC
instead of CAC to generate
SB100 (H178F).
H178F means there is a
phenylalanine amino acid at
position 178. in the protein
sequence of the transposase
instead of histidine.
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Ok, here is an another topic.
In our earlier example, we digested
the mutated SB100 plasmids with
PstI that generated 3 fragments.
How can we make predictions about
fragment lenght ?
We can detect the number and the
position of the sites of restriction
endonucleases related to the
plasmid using the info panel.
Restriction endonucleases like PstI
can cut the double-strand of the
plasmid DNA within a specific (and
usually) short sequence. It leads to
the enzymatic digestion of the
plasmid DNA.
Using the info panel, we can see
that this plasmid contains 3 sites of
PstI, the position of these sites and
specific sequence of PstI.
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How can we
perform this „digital
digestion” of the
plasmid ?
Analyses
Restriction analysesRestriction
fragments
Go to the next
page…
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You can choose PstI
from the list along
with other enzymes.
Go to the next page..
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And the information about
the lenght of the fragments
appeared:
- 4668 bp
- 1539 bp
- 433 bp
(The entire plasmid is 6640
bp)
1 kb
ladder
4668 bp
1539 bp
433 bp
The result of PstI digestion of the plasmid on
agarose gel. This is the expected pattern.
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