Site-directed mutagenesis using vector pUC19M and mutant
Escherichia coli (mut S) host.
Ruchi Patel and Shailendra Rijal.
Department of chemistry and molecular sciences, Macquarie University, NSW, Australia.
ABSTRACT:
This report mainly focuses on inducing the desired change into a plasmid DNA, by molecular
technique “site-directed mutagenesis”. Mutagenesis was carried out on one of the mutant of
pUC19 that is pUC19M. The experiment also involves use of two primers that is mutagenesis
primer and selective primer along with the use of mutant host (mut S E. coli). T4 DNA
polymerase helps in extension while T4 DNA Ligase is involved in ligation. The plasmid
DNA was recovered and was subjected to digestion by restriction enzyme at the unique
sequence. Recombinant and non-recombinant colonies were identified by the blue-white
screening technique using plates containing X-gal and IPTG. Recombinants and nonrecombinants were blue and white colonies respectively.
INTRODUCTION:
Mutagenesis is a process involving modification of genetic information of organism which
ultimately results in mutation. During the first half of 20th century, mutagenesis was
developed as a science based on the work by Hermann Muller, Charlotte Auerbach and J. M.
Robson.[1]
Site directed mutagenesis is a molecular biology method widely used to investigate the
structure and biological activity of DNA, RNA and protein molecules. Editing of genome and
mutagenesis may be performed in vivo by using CRISPR/Cas9 technology[2].
Due to the advancement in synthesis of oligonucleotide primers it is possible to achieve the
specific targeted change in the double stranded DNA of plasmid vector. (Paul cartyer, 1985).
The experimental procedure involves the use of two oligonucleotides that is mutagenesis
primer and selective primer. [4] Mutagenesis primer incorporates the desired change in the
given nucleotide sequence while, selection primer anneals the region containing unique site
and in which is incorporated a change designed to remove that restriction site. The selection
primer used for the experiment is 21 bases long and made to anneal to sequence surrounding
the unique Nde1 site in pUC19M at position 183. [3]
The extension of the single stranded DNA complementary to template strand was carried out
by using T4 DNA polymerase and later are transformed into E. coli cells deficient in mismatch repair. T4 DNA ligase joins the sticky ends and removes the nick which are created
during the hybrid formation.
The vector used for the experiment is a variant of pUC19 called pUC19M. The variant
contains selectable marker that is ampicillin resistance gene, multiple cloning site (MCS),
various restriction sites for different restriction endonucleases along and mutated Lac Z gene.
Identification of the transformants can be carried by blue-white screening procedure. For
screening, a chromogenic substrate known as X-gal is added to the agar plate. If βgalactosidase is produced, X-gal is hydrolyzed to form 5-bromo-4-chloro-indoxyl, which
spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’dichloro-indigo. The colonies formed by non-recombinant cells, therefore appear blue in
colour while the recombinant ones appear white. The desired recombinant colonies can be
easily picked and cultured. Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a nonmetabolizable analog of galactose that induces the expression of lacZ gene. [4]
In case of variant pUC19M, the reversion primer allows substitution of STOP codon by TRP
codon, thereby allowing complete translation the lacZ gene on X-gal plate. Hence, in this
experiment by site-directed mutagenesis we will be attempting to revert the mutation in
pUC19M to wild type. Therefore, the transformation results in the production of nonrecombinant white colonies and recombinant blue colonies containing wild type lac Z gene.
The aim of the experiment is to study the site-directed mutagenesis using mutated vector
PUC19M and attempting to revert the mutation in pUC19M to wild type.
METHODS AND MATERIALS:
Second strand synthesis:
pUC19M DNA was previously denatured by heating at 100ºC for 3 minutes, resulting in
single strand formation. Addition of 2µl each of annealing buffer, mutagenesis primer and
selection primer to the sample was carried out. Further, heated to 60ºC for 5 minutes, chilled
on ice and spin. Addition of T4 DNA polymerase, T4 DNA ligase, water and synthesis buffer
was carried out, mixed and incubated at 37ºC for 2 hours.
Transformation:
The reaction mixture containing DNA was diluted 5 fold with sterile water. 1µl of diluted
DNA was added to the E.coli BMH 71-18 mutS competent cells, incubated on ice for 15
minutes. The cells were heat shocked at 42ºC for 1 min to which L-Broth, nutrient media for
growth was added. Incubation along with shaking at 37ºC was performed for 25 minutes. Lbroth and ampicillin were added to the sample with final concentration of 50µg/ml and were
kept for shaking overnight at 37ºC.
Isolation of Plasmid DNA:
Plasmid DNA was isolated using various solution such as cell lysis solution, neutralization
solution and alkaline protease solution. 10µl of plasmid DNA was digested with Nde1. Both
digested and non-digested DNA were purified by spin column chromatography.
Final Transformation:
Late log phase JM101 cells were centrifuged and cells were recovered in CaCl2 and
resuspended, then re-spin. Both the samples that is sample containing Nde1 digested cells and
non-digested cells were heat shocked and then plated onto the plates containing ampicillin,
X-gal and IPTG.
RESULT:
Site directed mutagenesis was carried out using the original non-digested Nde1 plasmid and
digested Nde1 plasmid. The plates containing no digested DNA showed growth of white
colonies with tiny blue colonies at the background; while the plates containing Nde1 digested
DNA showed the presence of both blue and white colonies.
The following table shows the number of colonies obtained on each plate by our group:
NO DIGEST
Nde1 Digest
White colonies
Blue colonies
White colonies
Blue colonies
106
0
88
33
The following is the plate for our group RP/SR with Nde1 digested DNA:
The following is the plate for our group RP/SR with no digested DNA:
The following table shows the class data of total number of colonies obtained from class A
and B respectively.
No digest
Blue colonies
Nde1 Digest
White colonies
Blue colonies
White
colonies
CLASS A
11
1136
425
986
CLASS B
26
911
436
866
Total Number of
37
2047
861
1852
colonies
The percentage of blue colonies obtained on the plate containing no digested DNA was found
to be 1.77% while the percentage of blue colonies obtained on the plate containing Nde1
digested DNA was found to be 31.73%
DISCUSSION:
The method used for transformation is chemical method using calcium chloride. Use of this
chemical makes the cells membrane leaky by increasing the permeability of membrane and
helping intake of the plasmid DNA. Other methods which can also be used for transformation
are physical methods such as electroporation, biolistics, agitation with glass beads,
ultrasound, shock-waves etc. however the efficiency in this method is also very less and also
the risk of DNA damage is more.[5]
The white colonies obtained on the plate containing no digested DNA were due to activated
mutant Lac Z gene. The blue colonies were due to recombination of DNA on X-gal plate.
Complete translation of mutant lac Z gene was observed as STOP codon was substituted by
TRP codon at the recognition sequence of Nde1. From the results obtained it can also be
concluded that the mutagenesis was successful.
A lawn of tiny blue colonies was also observed along with the white colonies in case of plate
containing no digested DNA it may be due to wrong ampicillin concentration probably less
concentration.
The transformation frequency of blue colonies was increased by approximately 18 folds,
however the transformation frequency should be 70% which was not observed. The possible
reasons for it can be lower technique used was not good. To obtain proper results the final
step of transformation should be done again.
In conclusion, site-directed mutagenesis was successfully achieved along with reversion of
mutation from pUC19M to wild type.
1. 5’phosphorylated end will provide site for ligation of DNA. The procedure involves use of
restriction enzymes and hence the fragments generated with the sticky ends can only be
ligated to the host DNA by T4 ligase only if it recognises the 5’ end.
2. It is very important that the host in first transformation should be mut S mutant which is
deficient in mismatch repair. Since, the non-deficient will remove the substituted nucleotide
as part of repair mechanism. Second transformation involves, blue white screening to
determine the non-recombinants and recombinants. Hence, in second transformation it is not
necessary that the host should be mut S.
3.The plate containing X-gal shows the presence of blue colonies indicating change in the
recognition sequence from “CATATG” to “CAAATG” when digested with Nde1. The
selection primer used will help to recognize the unique sequence which is present at the Nde1
site. The restriction endonuclease Nde1 becomes non-functional due to mutation which
replaces nucleotide T to A and hence the DNA strands formed cannot be cleaved.
4. The features of the plasmid include that they should be complementary to the DNA strand
and should be 5’-phosphorylated to ensure proper ligation. The selection primer should be
few base pairs long to recognize the unique restriction site to bind.
BIBLIOGRAPHY:
[1] Beale, G. (1993). "The Discovery of Mustard Gas Mutagenesis by Auerbach and Robson
in 1941". Genetics. 134 (2): 393–399.
[2] Hsu PD, Lander ES, Zhang F (June 2014). "Development and applications of CRISPRCas9 for genome engineering". Cell. 157 (6): 1262–78. doi:10.1016/j.cell.2014.05.01
[3] Deng, W.P. and Nickoloff, J.A. (1992). Site-directed mutagenesis of virtually any plasmid
by eliminating a unique restriction site. Analytical Biochemistry 200: 81-88.
[4] Carter, P. (1987) in Methods in Enzymology (Wu, R., and Grossman, L., Eds.), Vol. 154,
pp. 382-403, Academic Press, San Diego.
Some website resources:
[5] http://www.sigmaaldrich.com/technical-documents/articles/biology/blue-whitescreening.html