TITLE: Mutant generation in Streptococcus mitis strain B6
AUTHORS: Samantha King

[DESCRIPTION]
This protocol is the methodology that we have successfully employed to generate and confirm insertion deletion mutants in Streptococcus mitis strain B6. Attached to the protocol is a file that includes primers for the MonX mutation

[STEPS]
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.       
Creation of a plasmid construct
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.1. 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.  
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.2. The spectinomycin resistance cassette (aad9) was amplified using
primers S1 and S2 
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.3. PCR products were then purified with a Qiagen PCR Purification Kit  
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.4. The three fragments for each mutant construct were cloned into pJET 1.2/Blunt (Thermo Fisher Scientific) using In-Fusion Snap Assembly (Takara) and transformed into Escherichia coli stellar competent cells. 
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.5. Transformants were selected on LB agar plates supplemented with ampicillin (100 µg/ml) and incubated at 37oC overnight
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.6. The resulting colonies were confirmed as ampicillin resistant by streaking on a new LB agar plate supplemented with ampicillin (100 µg/ml).
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.7.  Transformants were screened by colony PCR using pJET 1.2/blunt Fwd and Rev primers.
SECTION: Streptococcus mitis B6 mutagenesis protocol
2. Transformation of S. mitis B6
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.8. 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.
SECTION: Streptococcus mitis B6 mutagenesis protocol
1.9. The plasmid was then purified using Qiagen Miniprep Kit and confirmed by sequencing
SECTION: Streptococcus mitis B6 mutagenesis protocol
2.1. 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).
SECTION: Streptococcus mitis B6 mutagenesis protocol
2.2. 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) (EMRKPDGALFNLFRRR - 1 mg/ml), and 150 ng of DNA. A no DNA control tube is included to account for potential contamination.  These tubes were prewarmed in a waterbath to 37°C.
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2.3. When the culture reached an OD600 of 0.10 to 0.12, 50 μl of culture was added to the prewarmed tubes.
SECTION: Streptococcus mitis B6 mutagenesis protocol
2.4. Tubes were incubated in a waterbath at 37 °C for 2 hr. 
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2.5. 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).
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2.6. Plates were incubated at 37oC in 5% CO2 overnight and then patched onto selective plates.
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3. Confirmation of putative transformants
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3.1.  Putative transformants were grown in tryptic soy broth and DNA prepared as previously described [2]. 
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3.2. The mutations were confirmed by PCR and sequencing (using primers S1 and S2) or genome sequencing.