Gene Cloning

Screening DNA Libraries 137
ized expression vector (Section 9.3) to ensure that the protein is expressed at
high enough levels to be detected.
Q5.4. Describe the approach you would use to clone the gene for (a) β-
galactosidase (see Section 3.9) from the Gram negative bacterium
Citrobacter, and (b) human insulin. You should consider what type of DNA
library would be the most suitable and what form of screening for expression
you could use.
A5.4. (a) With a bacterial gene, like the gene for β-galactosidase, a genomic
library would be your best option. It is probable that the gene from
Citrobacter would be expressed in E. coli but you may choose to use a plasmid
expression vector to make sure that the gene is expressed at significant
levels in E. coli. Complementation of a lacZ mutant of E. coli should work
here as Citrobacter and E. coli are closely related, but you could also look for
direct expression of β-galactosidase using X-gal and IPTG. In any case you
will still need to use a lac – strain of E. coli to eliminate background expression
of β-galactosidase.
(b) With a human gene you will probably want to make a cDNA library, the
pancreas would be a good source of cells with insulin mRNA. You will have
to use immunological screening so you will probably use a phage vector as
phage lysis will release proteins from the cells making screening easier. You
will need to raise an antibody to insulin, which means that you will need a
pure sample of the protein; again this can be prepared from pancreas tissue.
As you are using immunological screening you will need to be sure that the
insulin is expressed in the E. coli host, you will need to make your cDNA
library in a specialized expression vector.
Q5.5. Why is it not possible to derive an unambiguous DNA sequence from
the amino acid sequence of a protein?
A5.5. Because of the degeneracy of the genetic code any given amino acid
may be encoded by up to six different triplet codons. For example, leucine is
encoded by CUU, CUC, CUA, CUG, UUA and UUG (Box 8.1).
Q5.6. What factors would be important in deciding how long an oligo
probe needs to be?
A5.6. The considerations are the same as with designing oligonucleotide
primers for PCR (Box 3.6). Probes need to be at least 17 bp long to ensure that
the sequence will not occur by chance. They are not usually more than about
25 nucleotides long, partly because of expense but also because of the problems
created by the degeneracy of the genetic code. As with PCR primers the
melting temperature is an important consideration (Section 5.8).