Mutant generation in Streptococcus oralis strain S.mitis/oralis 351

Creation of a plasmid construct t

The regions upstream and downstream of the region to be deleted were amplified

using primers 1 and 2, and 3 and 4, respectively. These primers were designed

to contain appropriate overhangs to allow In-fusion with pJET 1.2/blunt and the

antibiotic resistance cassette.  

The spectinomycin resistance cassette ( aad 9) was amplified using

primers S1 and S2

PCR products were then purified with a Qiagen PCR Purification Kit

The three fragments for each mutant construct were cloned into pDRIVE using In-Fusion Snap Assembly (Takara) and transformed into Escherichia coli stellar competent cells.

Transformants were selected on LB agar plates supplemented with ampicillin (100 µg/ml) and incubated at 37^oC overnight

The resulting colonies were confirmed as ampicillin resistant by streaking on a new LB agar plate supplemented with ampicillin (100 µg/ml).

Transformants were screened by colony PCR using M13 Forward and Reverse primers.

For transformants giving an appropriate PCR product, a 5 mL LB culture supplemented with ampicillin (100 µg/ml) was grown overnight at 37 °C with shaking at 200 rpm.

The plasmid was then purified using Qiagen Miniprep Kit and confirmed by sequencing

Transformation of S. mitis B6 S. mitis B6

Strains were growth at 37°C in C+Y pH8 [1] – starting at a low inoculum i.e. from a plate or diluting from a culture 1:100 (starting optical density at 600nm [OD₆₀₀] = 0.03 to 0.05).

When the culture was close to OD₆₀₀ =0.1, 950 μl of C+Y pH 8.0 medium was added to a 1.5 ml tube with 10 μl of 100 mM CaCl2, 2 μl of competence stimulating peptide (CSP) ( DKRLPYFFKHLFSNRTK - 1 mg/ml), and 150 ng of DNA. A no DNA control tube is included as a negative control.  These tubes were prewarmed in a waterbath to 37°C.

When the culture reached an OD₆₀₀ of 0.12, 50 μl of culture was added to the prewarmed tubes.

Tubes were incubated in a waterbath at 37 °C for 2 hr.

Reactions were pelleted by centrifugation and resuspended in approximately 100 µl of media. This was plated on selective Tryptic Soy Agar (TSA) plates spread with 5000 U catalase (Worthington Biochemical Corporation).

Plates were incubated at 37oC in 5% CO2 overnight and then patched onto selective plates.

Confirmation of putative transformants

Putative transformants were grown in tryptic soy broth and DNA prepared as previously described [2].

The mutations were confirmed by PCR and sequencing (using primers 5 and 6). Genetic background was confirmed by repetitive extragenic palindromic PCR [3].