Summary Practicals Modules Gene Technology

Module 1
Experiment 1: Mutant complementation

1. Genotyping of a mutant E. coli for absence of an amino acid biosynthetic gene
2. PCR amplification of the wild type gene from E. coli genomic DNA
3. Classical cloning of the wild type gene in an expression vector
4. Golden Gate cloning of the wild type gene as a modular part and creating a
transcriptional expression unit
5. PCR amplification and Golden Gate cloning of an orthologous plant gene as a
modular part and creating a transcriptional expression unit
6. Transformation and testing successful cloning as well as mutant complementation

Experiment 2: CRISPR/Cas9 assisted gene editing
1. Golden Gate cloning of an sgRNA protospacer
2. Generate mutations in E. coli by induced CRISPR/Cas9 gene editing
3. Confirm induced mutation by selection and allele specific genotyping PCR

E. coli strain (JW3841) that through site-directed recombination is mutated in a gene which
has made this strain deficient for biosynthesis of one particular amino acid (auxotroph is
another word for this type of phenotype)  this amino acid must be present in the medium
for the E.coli to survive.

1. Genotyping of a mutant E. coli for absence of an
amino acid biosynthetic gene
Final proof that the mutated gene is the cause of the observed phenotype is obtained by
insertion of a working copy of the original gene (complementation). The complementation
experiment means that the gene needs to be provided in a genetic construct under the
control of working promoter and in the presence of transcriptional terminator sequence.

JW3841 is not able to synthesize its own glutamine. In media without glutamine, this strain
will not be able to grow. To prove that this phenotype is due to the glnA mutation, you will
try to complement this mutation by introducing a plasmid expressing glnA in JW3841.

The KEIO collection as tool to study gene function in E. coli
One solution to the problem of immediate mutant allele access for any gene is the creation
of very large collections of (sequence-indexed) insertion lines for an organism.

® Mutant from the Keio collection is generated by gene-specific knock-out through
homologous recombination
o the specific target gene is replaced by a gene conferring resistance to the
antibiotic kanamycin.

, ® Operon mutations may prevent complementation by single gene addition because
they can affect all genes in the operon and we would need to introduce multiple
genes

Genotyping
The glnA gene encodes for the protein glutamine synthetase. This enzyme catalyzes the
ATP-dependent biosynthesis of glutamine from glutamate and ammonia as such:




Generally genotyping by PCR can be used to detect sequence variations in alleles or insertion
of transgenes in specific cells or organisms. The primer sets are designed to flank regions of
interest and assess genetic variations based on the presence or absence of an amplicon
and/or its length
® To prove that the Kan recombination indeed has taken place in glnA, you need to find
primers within the flanking sequences, that in combination with primers located in
the Kan and glnA gene can be used in a genotyping PCR.




Steps order:

, 1. Get WT E. coli and mutant E. coli: single colonies on an agar plate, or precultures
2. Grow both strains
3. Isolate genomic DNAs
4. Measure the DNA concentrations
5. PCR on both DNAs using genotyping primers
6. Run samples in a gel



2. Conventional cloning
Plasmid vector: pBluesScript (pBS) that has a multiple cloning site (MCS)
 In the presence of the substrate X-gal this gives blue colonies when no insert is
present in the MCS in pBS.
 use the LacZ promoter to drive expression of glnA gene.
o use two extended primers that contain a different restriction site at their 5’,
XhoI and HindIII.
 Following PCR amplification, you should produce a glnA amplicon with a XhoI site at
the 5' end and a HindIII site at the 3' end to allow in frame cloning of glnA just behind
the lacZ start codon. This glnA containing XhoI-HindIII amplicon is generated by PCR
on wild-type BW25113 genomic DNA
 The amplicon is cut using XhoI and HindIII enzymes, and the large amplicon fragment
with XhoI and HindIII ends (the blue part in the picture below) is used as an insert in
the ligation reaction mixture. The pBS cloning vector will of course be digested with
the same enzymes.
 Subsequently the glnA coding sequence will be ligated in the MCS of pBS. From this
recombinant plasmid, a functional glnA protein will be expressed and this plasmid
will therefore also be used for complementation tests of the JW3841 mutant.


Steps glnA PCR:
1. Get genomic DNA from wild-type E.coli strain BW
2. Measure the concentration of the DNA
3. PCR amplify glnA using extended primers (that contain a different restriction site at
their 5’, XhoI and HindIII) to allow in frame cloning
4. Run a gel
5. Purify DNA fragments
6. Measure the concentration of the glnA amplicon
a. This glnA containing XhoI-HindIII amplicon is generated by PCR on wild-type
BW25113 genomic DNA

Vector pBSKS(+) is cut using HindIII and XhoI enzymes, and the larger fragment is used in the
conventional cloning of glnA.

Steps conventional cloning of glnA from WT E. coli:
1. Digest the amplicon to generate the insert fragment having overhanging ends; digest
plasmid pBlueScript to generate the vector fragment having the same overhanging
ends