Antibiotic Resistance Protocols (Methods in Molecular Biology)

18

their ease of transformation into bacterial cells, but are unfavorable

for in vivo experiments due to the propensity for loss of the plas-

mid when not maintained by selective pressure. Furthermore, dif-

ferences in plasmid propagation and copy number may introduce

unintentional experimental bias. It is therefore preferable to inte-

grate the antibiotic resistance marker into the chromosome.

Whilst experiments are traditionally carried out in the absence

of antibiotic treatment of the host organism, one key advantage of

using resistance markers for quantification is the additional ability

to treat the infected host with the relevant antibiotics in order to

study the effect of chemotherapeutic intervention on drug- resistant

bacteria. The response of the invading pathogen to subcurative

doses, such as those that might be encountered during ineffective

or incomplete treatment, is of particular interest. The strains

described in this protocol have been successfully used in such

experiments [ 4 ].

2 Materials

Use ultrapure deionized water (resistivity 18 MΩ cm at 25 °C)

during all steps. Unless otherwise stated, store solutions and mate-

rials at room temperature. DNA solutions should be stored at

− 20 °C. Standard molecular cloning techniques and kits should be

used; where our methods differ from manufacturers’ protocols, the

modified methods are explained in full. This protocol assumes

basic molecular biology knowledge and equipment/reagents suit-

able for cloning DNA in E. coli only. Molecular biology methods

for working with S. aureus are given in detail where appropriate.

Where filter sterilization is mentioned, use 0.45 μm pore size

membranes.

1. Oligonucleotide primers (see Subheading 3.1 and Note 1).


2. Purified genomic DNA (or a fresh colony) of the target organ-
   ism (see Note 2).


3. Replication-permissive cells containing a suitable suicide vec-
   tor (see Note 3).


4. Preferred high-fidelity PCR reagents.


5. Preferred DNA ligase.


6. Preferred linear DNA and plasmid purification (“miniprep”)
   kits.


7. Agarose and equipment for gel purification of DNA.


8. Standard equipment and reagents for heat shock or electro-
   poration of E. coli.

2.1 Strain
Construction: Suicide
Vectors

Gareth McVicker et al.