PCR

Purpose: to check whether our plasmid has successfully transformed into E. coli, DH5α.

• 1. PCR condition finding

•   * P.S. Prepare the stock solution first, which consist of mQH₂O、10x buffer、forward primer、backward primer、dNTP and DNA, then add the DNA polymerase before starting the PCR process.
• 0.5~1 tube shall be added to the ideal amount when preparing the stock solution.
• It is recommended to dilute the amount polymerase needed to 1 unit/ μl, to decrease the error of adding small amount of volume.

Table 2. Recipe for PCR.

Table 3. Final concertation for PCR.

• 2. Condition entering

• a. 94°C for 1 min.
• b. 94°C for 30 sec
• c. The specific temperature for primer to bind on DNA, 30 sec (pSBBS1C 55°C, pSBBS4S 65°C)
• d. 72°C for 1 min
• e. GOTO 2, 25 cycle
• f. 16°C for 10 min (to cool the machine)
• g. Run gel
• h. Results

B. subtilis Competent Cell Preparation and Transformation

• 1. Streak recipient strain on LB Agar plate. Incubate overnight (18 hour) at 37°C.
• 2. Inoculate a few colonies into 4.5 ml of Medium A in a 16×125 mm test tube that lacks visible scratches. Mix the contents of the tube thoroughly. Read its optical density at 600 nm in the spectrophotometer. Adjust the OD₆₀₀ to be 0.1-0.2, maintaining the volume at 4.5 ml.
• 3. Incubate at 37°C with vigorous aeration. Read the OD₆₀₀ every 20 min, plotting OD₆₀₀ against time on semi-log paper. After a brief lag, the OD should increase logarithmically—that is, they should fall on a straight line. Note the point at the culture leaves log growth—the graph points fall below the straight line. In B. subtilis genetics, this point is known as t₀. It should take 60~90 minutes of incubation and occur at OD₆₀₀=0.4-0.6.
• 4. Continue incubation for 90 minutes after the cessation of log growth (t₉₀). Transfer 0.05 ml of this culture into 0.45 ml of pre-warmed Medium B in a 16×125 mm test tube. Set up one tube for each transformation you intend to perform, plus an extra for a DNA-less control (negative control).
• 5. Incubate the diluted cultures at 37°C with vigorous aeration for 90 min. At this point, the cultures should be highly competent.
• 6. Add 1 µg of DNA to the competent cells and incubate at 37°C with aeration for 30 minutes.
• 7. Plate 200μl of the transformed cells onto selective agar.

Table 4. Recipe of 10× Medium A base.

Autoclave, then add: 50% glucose, filter-sterilized 100 ml

Table 5. Recipe of 10× Bacillus salts.

Add ddwater to 1000 ml, autoclave 121°C, 1.2 atm for 60 minutes.

Table 6: Recipe of Medium A.

Table 7. Recipe of Medium B.

*Need to add threonine (5 mg/ml) for strains carrying an insertion in thrC, 100μl/10 ml medium.

Results

Reference

• 1. Bacillus Genetic Stock Center Catalog of Strains, Seventh Edition Volume 4: Integration Vectors for Gram-Positive Organisms, page 17
• 2. LMU-Munich_2012_Bacillus_subtilis_vectors

Lactate sensor Function test

As we mention before, we what to evaluate the risk of caries by detecting the three parameters in saliva: concentration of CSP (competence-stimulating peptide), lactate level and pH value.
About our lactate sensor, the function what we need is not only detect lactate, but also quantize it. So we did this experiment to insμre that the GFP light intensity as different as lactate level.

• 1. Pre-culture in 15 ml test tube
• Culture bacteria (E. coli-DH5α,pSB1A2-O1-J23118-O2-GFP-J23118-LldR) in 5 ml LB + 5 μl Amp^R (100 μg/ ml, final), incubate in 37°C 14 hour.
• Also pre-culture the bacteria that don't have fluoresces gene as negative control (E. coli-DH5α,pUCIDT-kan^R –pBAD-mazEF).
• 2. Refresh in 250 ml Erlenmeyer flask
• 20 ml LB + 400 μl pre-culture bacteria + 20 μl Amp^R (100 μg/ml, final), 37°C, 170 rpm incubate for 90 min. Also refresh negative control.
• 3. Transfer bacteria to 96 well plate.

Table 8. Recipe of lactate sensor function test.

• 4. Add three glass beads for each well.

Table 9. Concentration of lactate in lactate sensor function test.

• 5. After finish loading plate, put them into incubator and fix well, 37°C, 170 rpm.

Fig 2. 96 well plate for fluorescence.

Fig 3. 96 well plate forOD₆₀₀.

• (We didn't take a photo for our experimental plate. This plate is just for example, that is water in those well.)


• 6. Measurement:
• Each 15 min, measure the fluorescence and OD₆₀₀ by plate reader and Elisa reader respectively.

Functional test for Bacillus subtilis

Supposedly, GFP should appear when competence stimulating peptide (CSP) was added. To make sure this function works properly in Bacillus subtilis, we've designed a functional test for it.

• 1. The transformed Bacillus subtilis which contains the target genes we need was incubated in LB medium at 25°C, overnight.
• 2. Measure the OD₆₀₀, then dilute the bacterial culture to 0.4, 0.2, 0.1, and 0.05 in the value of OD₆₀₀.
• 3. Dilute CSP into 4 different concentration – 1 mg/ml, 500 g/ml, 250 g/ml, 125 g/ml
• 4. Load the mixture above into a black microplate and a clear microplate as the picture shown below.
• 5. Measure the clear plate with the absorbance of wavelength 595 nm, and the black plate with a fluorescent plate reader (excitation: 485 nm, emission: 520 nm ) every 15 minutes.
• 6. Results

Fig 4. 96 well plate for B. subtilis function test.

For G1 ~ G3: Bacterial culture OD₆₀₀ = 0.4, For G4 ~ G6: Bacterial culture OD₆₀₀ = 0.2, For G7 ~ G9: Bacterial culture OD₆₀₀ = 0.1, For G10 ~ G12: Bacterial culture OD₆₀₀ = 0.05, CSP-free for line G and H