Streptococcus mutans is naturally competent, able to uptake exogenous DNA when its competence pathways are induced by quorum sensing signals. These signals can be provided artificially in the form of competence stimulating peptide (CSP) in rich media or as comX inducing peptide (XIP) in minimal media. This protocol describes how to induce transformation of our strain of *S. mutans*, JH1140 by using horse serum and CSP in a Todd Hewitt and yeast extract media. ## Materials - [ ] Viable *S. mutans* colonies of your desired genotype(s) - [ ] Filter sterilized aqueous 200 µM CSP-21; 8 µL per experimental condition or replicate [^1] - [ ] [[THYE#THYE + horse serum (HS)|THYE + horse serum]]; ~20 mL per overnight culture - [ ] [[THYE]] broth for serial dilutions.[^2] - [ ] [[THYE]] plates with and without your selectable marker.[^2] - [ ] 15 mL screw cap conical tubes, 1 per overnight culture - [ ] 250+ ng of donor DNA per experimental condition or replicate - [ ] Sterile 96 well plates[^3] - [ ] 200 µL capacity and 20 µL capacity multichannel pipettes and compatible tips - [ ] Sterile reservoir ## Planning your 96-well plate and overnight cultures Each **row** of your 96-well plate corresponds to one unique condition or replicate, with two rows for a positive and negative control with predictable outcomes. Always choose control conditions that you have experimentally verified. For example: | Row | Genotype | CSP | DNA | Replicate | | :-: | :------: | :--: | :------------------------------------------: | :---------: | | A | WT | 8 µM | None | *– control* | | B | WT | 8 µM | 250 ng of donor DNA with a resistance marker | *+ control* | | C | *∆mutA'* | 8 µM | 250 ng of donor DNA with a resistance marker | 1 | | D | *∆mutA'* | 8 µM | 250 ng of donor DNA with a resistance marker | 2 | | E | *∆mutA'* | 8 µM | 250 ng of donor DNA with a resistance marker | 3 | | F | *∆mutA'* | 0 µM | 250 ng of donor DNA with a resistance marker | 1 | | G | *∆mutA'* | 0 µM | 250 ng of donor DNA with a resistance marker | 2 | | H | *∆mutA'* | 0 µM | 250 ng of donor DNA with a resistance marker | 3 | Each **column** will correspond to one 10-fold dilution step, where column 1 is your undiluted culture, column 2 corresponds to $10^{-1}$, column 3 to $10^{-2}$, and so on through column 9 at $10^{-8}$. Each **replicate** of the same experimental conditions should have a unique overnight. Independent experimental conditions with the same genotype may share an overnight – this can, under some circumstances, allow you to perform paired T-tests, increasing statistical power. In this example, you need a minimum of four overnights – one WT, and three *∆mutA'*. ## Prep work - [ ] Use a sharpie to label the lid of the 96-well plate with the following information in advance: - Each **row** should be labeled according to the planned experimental condition - Each **column** should be marked according to the dilution step; -1, -2, etc. - [ ] Fill the serial 96-well plate dilution columns $10^{-1}$ to $10^{-8}$ with 180 µL of standard [[THYE]] in each well. Remember to leave the first column empty. Parafilm and store at 4ºC. - [ ] Aliquot 10 mL of [[THYE#THYE + horse serum (HS)|THYE + HS]] (0.2% YE w/v) into 15 mL screwcap conical tubes for as many conditions or replicates as you wish to test. Store at 4ºC. - [ ] Set aside a portion of the same batch of media to use as a blank for the spectrophotometer and to dilute the culture(s). Store at 4°C. - [ ] Label one plate with the selectable marker and one without per row in your 96-well plate. - Dilution columns 1 through 5 ($10^0$ through $10^{-4}$) should be plated on media with your selectable marker to isolate and count transformed cells. - Dilution columns 5 through 9 ($10^{-4}$ through $10^{-8}$) should be plated on media without your selectable marker to count viable cells. ## Transformation protocol 1. The afternoon before the transformation, set your labeled plates out at room temperature to dry overnight to reduce spot merging during spot assay plating. 2. Inoculate each overnight with a colony of *S. mutans* and incubate overnight (~16 hours) at 37°C, shaking at 250 rpm. Set the needed blanking and dilution media out at room temperature overnight. 3. In the morning measure the $OD _{600}$ and dilute each culture to a final $OD _{600}$ = 0.1.[^4] 4. Using the same incubation conditions, grow the culture for an additional 60 to 90 minutes, measuring at 60, 75, and 90 minutes. 5. When the culture reaches $OD _{600}$ = 0.2 ± 0.02, aliquot 200 µL of culture into column 1 of each row of your 96-well plate according to your planned conditions.[^5] 6. To each 200 µL aliquot, determine the needed volume of purified donor DNA (default 250 ng equivalent volume) **and** CSP (default 8 µM). Waste that total amount of volume from each culture first, then add the required DNA and CSP so the final volume remains 200 µL per well. 7. Place the 96-well plate in a standing incubator at 37°C *without shaking* for 90 minutes.[^6] 8. After the 90 minutes have passed, dilute cells using a multi-channel pipette to transfer 20 µL from each column moving left to right, starting in column 1. Suspend cells by pipetting before each transfer. 9. Once cells are diluted, use a multichannel pipette to perform spot assays on an appropriate combination of selective and non-selective media for enumeration.[^7] [^1]: The amount required will vary depending on the CSP concentrations you intend to use to induce competence. For this protocol, we assume you are operating at an 8 µM final concentration of CSP. We also assume you are using synthetic CSP, but there should be no functional difference between naturally derived and synthesized. [^2]: Required volume or count depends on enumeration strategy; spread plating, spot assay, etc. [^3]: This protocol assumes you will be using a 96 well plate. However, you may substitute 96 well plates with an equivalent arrangement of microcentrifuge tubes if desired. [^4]: If the culture appears extremely turbid to the naked eye, you may save time by performing a 10-fold dilution before the first OD measurement. If this dilution is too extreme, simply revert back to using the original overnight culture tube. [^5]: If your culture has grown outside this range, you must re-dilute to 0.1 and outgrow again. [^6]: If plates for serial dilution plating have not already been labeled, this is a good time to do so. [^7]: 6 rows of 9 µL spots per plate works well to achieve a total of 54 µL plated per condition with reduced merging of spots, as compared to 5 rows of 10 µL.