Experiment
General protocol
- Transformation ( by 1.5 ml Eppendorf tube)
1. 2.5 μg DNA + 25 μl competent cell (ECOS 101 DH5α)
2. On ice 30 min
3. 42℃ heat shock 30 sec
4. On ice 5 min
(In laminar flow)
5. Add LB to 500 μl，37℃ 170 rpm incubate 2hour.
6. plate 200 μl ( 300 μl remained) spread over plate by spreader.
7. Incubate the plate in 37℃ overnight.
Patch and small scale culture
1. Pick a colony by toothpick then “patch” on another agar plate as bacteria stock. Incubate in 37℃ 14 hour~16 hour.
EX:
2. Dip the toothpick into 5 ml LB (with antibiotic) as small scale culture for plasmid extraction. Incubate in 37℃ 170rpm for 14 hour~16 hour.
Mini extraction
Follow the protocol of commercial gel extraction kit.
Digestion
1. 0.5 μg DNA + 0.2 μl fast digestion restriction enzyme ( 10 unit/μl, final 0.25 μg/unit) + 1 μl 10X buffer + mQ H2O -> total volume 10 μl.
2. Bath in 37℃ warm water for 15 min.
Ligation protocol
*The ideal volume for DNA ligation is 10 μl, but it is adding the volume to 15~20 μl is fine, since part of the liquid will vaporize during ligation.
*It is recommended that the concentration for the backbone is 20 fmol.
*The molar ratio for backbone : insert = 1 : 3
*The ligase required 0.1 ~ 0.5 unit/tube.
1. Quantify the concentration of the linear DNAs that you are going to ligate.
2. Calculate the molar weight of DNA. Converse the unit into ng/fmol.
3. Calculate how much Nano gram is required for 20 fmol of backbone and 60 fmol of insert.
4. Then by the quantified concentration, find out that how much μl is required.
5. Adjust the total volume to 8.9 μl/tube.
6. Add 1 μl of 10x ligation buffer and 0.1 μl of T4 ligase (5 unit/μl)
7. Place it in 16°C water bath, overnight.
Run electrophoresis gel
1. X (g) agarose + Y (ml) 0.5XTAE make a suitable agarose gel( depend on your DNA size), X/Y = 1.2%~1.5% (<0.5k) heating until all agarose melted.
0.8%~1% (0.5~10k)
0.3%~0.7% (>10k)
Cool down the agarose gel (use the back of your hand to check it, as long as it does not burn your hand, it is suitable temperature) + EtBr ( final, 5 μl/100 ml).
2. Recipe:
| Marker | Sample | |
|---|---|---|
| Suitable ladder | DNA | |
| 2 μl | 10 μl | |
| 6X dye | 2 μl | 2 μl |
| mQ H2O | 8 μl | 0 |
| Total volume | 12 μl | 12 μl |
3. Load 12 μl per well, then run gel in electrophoresis tank under 100 V.
PCR
Purpose: to check whether our plasmid has successfμlly 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/l, to decrease the error of adding small amount of volume.
Recipe
| DNA (10 ng) | 10 ng |
|---|---|
| 10x buffer | 5 μl |
| forward primer (2.5 μM) | 2.5 μl |
| Reverse primer (2.5 μM) | 2.5 μl |
| dNTP (10 μM) | 1 μl |
| DNA polymerase (5 unit/μl) | 1~2 unit |
| Total volume , add water to 50 μl | 50 μl |
| Final concentration | |
|---|---|
| DNA | 10 ng/ 50 μl |
| Buffer | 1x |
| Forward primer | 0.125 μM |
| Backward primer | 0.125 μM |
| dNTP | 0.2 μM |
| DNA polymerase | 1~2 unit |
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. Result:
a. pSBBS1C
b. pSBBS4S
B. subtilis Competent Cell Preparation and Transformation
a. Streak recipient strain on LB Agar plate. Incubate overnight (18 hour) at 37°C.
b. 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.
c. 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.
d. 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).
e. Incubate the diluted cultures at 37°C with vigorous aeration for 90 min. At this point, the cultures should be highly competent.
f. Add 1 µg of DNA to the competent cells and incubate at 37°C with aeration for 30 minutes.
g. Plate 200μl of the transformed cells onto selective agar.
Recipe
10× Medium A base:
| Yeast extract | 10 g |
|---|---|
| Casamino acids | 2 g |
| Distilled water | 900 ml |
Autoclave, then add: 50% glucose, filter-sterilized 100 ml
10× Bacillus salts
| (NH4)2SO4 | 20 g |
|---|---|
| K2HPO4·P3H2O | 183 g |
| KH2PO4 | 60 g |
| Sodium citrate | 10 g |
| MgSO4·P7H2O | 2 g |
Add ddwater to 1000 ml, autoclave 121°C, 1.2 atm for 60 minutes.
Medium A
| Sterile water | 81 ml |
|---|---|
| 10× Medium A base | 10 ml |
| 10× Bacillus salts | 9 ml |
Medium B
| Medium A | 10 ml |
|---|---|
| 50 mM CaCl2·2H2O | 0.1 ml |
| 250 nM MgCl2·6H2O | 0.1 ml |
*Need to add threonine (5 mg/ml) for strains carrying an insertion in thrC, 100μl/10 ml medium.
1. Result
- Growth curve of Bacillus subtilis strain 168
- Transformation check for pSBBS1C
Since the amyE locus is knocked down, the transformed Bacillus subtilis won’t be able to digest starch. Hence when it is patched onto starch plate and placed overnight, the colonies shouldn’t have a bright orange surroundings when Lugol’s iodine is added.
Left: Bacillus subtilis strain MW12 without plasmid pSBBS1C
Right: Bacillus subtilis strain MW12 with plasmid pSBBS1C
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 AmpR(100 μg/ml, final), incubate in 37°C 14 hour.
Also pre-cμlture the bacteria that don’t have fluoresces gene as negative control (E.coli-DH5,pUCIDT-kanR –pBAD-mazEF).
2. Refresh: in 250 ml Erlenmeyer flask
20 ml LB + 400 μl pre-culture bacteria + 20 μl ampR (100 μg/ml, final), 37°C, 170 rpm incubate for 90 min. Also refresh negative control.
3. Transfer bacteria to 96 well plate:
| Final concentration of lactate | Refreshed bacteria(per well) | 1M lactate (per well) | mQ H2O (per well) |
|---|---|---|---|
| Positive control | 0.012 μg/ml fluorescein 200 μl | 20 μl | |
| Negative control | 200 μl | 0 μl | 20 μl |
| 0 mM | 0 μl | 20 μl | |
| 2 mM | 4 μl | 16 μl | |
| 4 mM | 8 μl | 12 μl | |
| 6 mM | 12 μl | 8 μl | |
| 8 mM | 16 μl | 4 μl | |
| 10 mM | 20 μl | 0 μl | |
| Blank | 200 μl LB | 0 μl | 20 μl |
4. Add three glass beads for each well.
| repeat | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9... | |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | A | positive | negative | 0 mM | 2 mM | 4 mM | 6 mM | 8 mM | 10 mM | blank |
| 2 | B | positive | negative | 0 mM | 2 mM | 4 mM | 6 mM | 8 mM | 10 mM | blank |
| 3 | C | positive | negative | 0 mM | 2 mM | 4 mM | 6 mM | 8 mM | 10 mM | blank |
5. After finish loading plate, put them into incubator and fix well, 37°C, 170rpm.
For fluorescence
For OD600
(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.
