Answers to examination in Gene technology, TFKE38 2012-10-20
1)
Inclusion body:
Aggregated protein that sometimes occurs when over expressed in E.coli
Tm value:
In molecular biology calculation of of melting temperature to decide optimal annealing
temperature in PCR.
Blue-white screening:
Use of Lac z-region for insertion and deactivate galactosidase function in the presence of
IPTG and Xgal. Colonies turns blue in the presence of an inserted fragment in the lacz gene.
Plaques:
Plaques are lysed bacteria formed on agar plates after infection with M13 or lambda phage.
Cosmid:
Syntethic construction of a plasmid containing cos sites an antibiotika resistence. Hybrid
between lambda phage and a plasmid.
2)
a)
Plasmid:
circular DNA, antibiotika resistant and needs competent cells for introduction into E.coli.
Phage:
Circular or linear DNA can insert often larger DNA fragment can directly infect E.coli.
b)
Plasmids: antibiotika selection, blue-white screening or colony screening
Phages: blue-white screening or colony screening
c)
Purification of plasmids: See fig 3.10 in Textbook page 34
Purification of phages: See Fig 3.23 in Textbook page 43
3)
a)
Alkaline phosphatase:
Removes the phosphate group at the 5’-end. Used to hinder relegation in gene cloning
experiments.
Terminal deoxynucleotide transferase:
Adds one or more deoxyribonucleotides to the 3’-end. Used to obtain sticky-emd in
combination with fragments amplified by taq polymerase (TA-cloning)
Polynucleotide kinase:
Reverse effect of alkaline phosphatase, adding a phosphate group at 5’-end. Used for labelling
with radioactive marker in e.g hybridization experiments.
b)
Use two different restriction enzymes with non-compatible sticky-ends. Treat vector with
alkaline phophatase to hinder relegation of the vector. Perform the ligation reaction at low
temperature (below room temperature)
4
a)
See fig 8.7 in Textbook page 134
b)
The constructed cDNA library display inserts of various size showing there is a variation in
the library.
c)
Forward primer:
5’-ATGGGGGTGCACGAATGTCCTGCC-3’
Reverse primer:
5’-TCAAAGGTCCCTGTCCTGCAGGGC-3’
d)
Change in the DNA sequence that do not cause any change in the amino acid sequence.
e)
A palindromic sequence:
CTTTGA change to 5’-CTATAG-3’ or 5’-TTATAA-5
3’-GATATC-5’ 3’-AATATT-3’
f)
The advantage is the possibility to regulate the transcription of the gene. If the gene product is
toxic and harmful for E.coli you can still express the protein at high yield.
g)
Codon bias:
There are different codon usage for E.coli compared to e.g human this cause a slow down in
the translation.
One way to avoid this by site-directed mutagenesis (if there only are a few sites) or
constructed a synthetic gene optimized for the E.coli codon usage.