How do you convert Bacillus subtilis?
Introduction Bacillus subtilis transformations can be done with complete plasmids as well as linear fragments. However, the type you choose depends on your specific experiment and desired result. If transforming with a shuttle vector, perform cloning in E. coli and isolate plasmid DNA.
How do you interpret transformation efficiency?
Transformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation procedure.
What is the doubling time of Bacillus subtilis?
120 min
The growth rate of individual cells of Bacillus subtilis (doubling time, 120 min) has been calculated by using a modification of the Collins-Richmond principle which allows the growth rate of mononucleate, binucleate, and septate cells to be calculated separately.
What is a good transformation efficiency pUC19?
Based on our experiments, maximal transformation efficiency for pUC19 was found to be 4.8×104 colony forming units per ug at 0.15 M of calcium chloride while pBR322 had a maximum transformation efficiency of 1.8×104 colony forming units per ug at 0.1 M of calcium chloride.
How do you make Bacillus subtilis competent cells?
- I thaw one aliquot of competent cell then inoculate in 20 ml LS medium.
- Shake cell at 150 rpm in 30 ºC water bath.
- Take 1 ml aliquot into eppendorf tube then add 10 μl of 0.1 M EGTA and incubate for 5 min at room temperature.
- Add plasmid pHT01 (2 μg) and incubate at 200 rpm for 2 h, 37 ºC.
Is Bacillus subtilis naturally competent?
Under certain growth conditions, Bacillus subtilis can develop natural competence, the state in which it is able to bind, adsorb and incorporate exogenous DNA. Development of competence is a bistable process and is subject to complex regulation.
How is transformation efficiency calculated pGLO?
- Transformation efficiency = Total number of colonies growing on the agar plate.
- Amount of DNA spread on the agar plate (in µg) Enter that number here Total number of colonies =
- (DNA in µg) = (concentration of DNA in µg/µl) x (volume of DNA in µl) Enter that number here.
- Total amount of pGLO DNA, µg.
How do you calculate transformation rate?
Usually people calculate the transformation efficiency (%) as: Transformation efficiency (%)= (Total number of PCR positive plants / Total number of inoculated callus) ×100.
What is the optimum temperature for Bacillus subtilis?
25-35 degrees Celsius
Bacillus subtilis is a spore forming, motile, rod-shaped, Gram-positive, facultative aerobe. It is mostly found in soil and vegetation with an optimal growth temperature from 25-35 degrees Celsius.
What is the elevation of Bacillus subtilis?
Table 2
| Bacterial isolates | Colour on nutrient agar | Elevation |
|---|---|---|
| Bacillus subtilis | White | Flat |
| B. cereus | Off-white | Convex |
| Escherichia coli | Mucoid | Slightly raised |
| Serratia | Mucoid | Umbonate |
What affects transformation efficiency?
The factors that affect transformation efficiency are the strain of bacteria, the bacterial colony’s phase of growth, the composition of the transformation mixture, and the size and state of the foreign DNA.
How do you make a transformation of Bacillus subtilis?
Transformation of Bacillus subtilis Transformation #1 Using fresh, competent B. subtilis cells: 1. Add 600 ng of DNA and incubate at 37°C (200 x g) for 1 h 2. Add 100 μl of expression mix to each sample 3. Incubate at 37°C for another hour 4. Plate 400 μl the samples on selective agar
What is the electroporation efficiency of Bacillus subtilis?
Electroporation efficiency for Bacillus subtilis regarding glycine concentration. The electroporation worked best with a higher glycine concentration. Glycine works as a weakening agent, making the cell wall looser by replacing the alanine in the cell wall [3].
Is it possible to clone Bacillus subtilis?
However, molecular cloning and transformation of B. subtilis are not as easy as those of Escherichia coli. Here we developed a simple protocol based on super-competent cells prepared from the recombinant B. subtilis strain SCK6 and multimeric plasmids generated by prolonged overlap extension-PCR.
How can I prepare OD600 for Bacillus subtilis?
Inoculate a liquid culture of Bacillus subtilis and let it grow overnight Vortex the culture gently and give 500 μl each in 3x 20 ml competency medium Grow the bacteria in the flasks at 37°C 250 rpm shaking till you reach an OD600 between 0.5 and 0.7/ml