Transformation of Gram-Positive Bacteria
an adaptation from Chang, D., Chassy, B., Saunders, J., Sowers, A. 1992.
Guide to Electroporation and Electrofusion, Academic Press, Inc., San Diego, CA.
Guiding principles: keep the cells as cold as possible at all times; manipulate the cells as gently as possible at all times.
1、Making electrocompetent gram positive bacteria
Start a fresh overnight culture with the bacteria of interest. Cells should be harvested at early to mid-exponential growth phase. Harvest the cells by centrifugation. Most strains should remain chilled during the entire procedure from the point of harvest.
Choose a growth medium for satisfactory growth under normal conditions. The medium may be optimized to yield the best efficiencies possible. Media defined as rich, limiting, defined, or minimal should be considered. Other variables affecting the growth of the bacteria may also considered such as temperature, substrates, and additions.
It is important to wash the cells thoroughly to remove any medium or electrolytes that may be present. Washing cells three to four times is adequate. The medium chosen to wash the cells and use as the electroporation buffer should 1) keep the cells viable, 2) have high electrical resistance. High quality deionized water works well in many applications.
Additives such as 0.27-1.0 M sucrose, 10-15% glycerol, 1 M sorbitol, PEG and similar reagents may be used to act as "osmotic stabilizers" or cryprotectants. In some instances, these additives help to increase efficiencies, in others, they interfer.
Buffers containing HEPES, MOPS, or phosphate are low resistance buffers and must be avoided. Magnesium and calcium chloride should also be avoided.
Resuspend the washed cells in electroporation medium to a final concentration of 5 x 10 exp 9 to 5 x 10 exp 10 cells/ml. Keep on ice until use. Many strains will remain electrocompetent for hours and can be frozen for future use. Quick freeze the cells in an dry ice bath and store at -70°C.
2、Electroporation of Cells
The pulse parameters should be made by consulting specific references for the genus and strain being transformed. If specific literature can not be found, evaluate the parameters for closely related species.
The controlling parameters are field strength (kV/cm) and time constant (tau , ms).
Typically, cocci will require higher field strengths (7 - 18 kV / cm) and the bacilli require lower field strengths. Preliminary experiments should be performed to determine the optimal field strength.
Optimal time constants for gram-positive bacteria are reported to be between 2.5 and 7.5 ms.
Mix the DNA of interest (0.1 - 1 μg in 1 - 2 μl of water) and the bacteria. Place the sample into a prechilled cuvette. Be certain the sample is in contact with both sides of the cuvette by tapping the cuvette lightly on a solid surface. Choose the cuvette size (1 or 2 mm) in accordance with the field strength amd sample volume desired. Sample size will be determined by the cuvette chosen. Insert the cuvette into the instrument and deliver the pulse.
Add media directly to the cuvette and plate aliquots on selective media. Incubate under normal conditions.
It may enhance transformation to allow the cells to recover in a non-selective media for a period of time (typically an hour) before plating. This allows the cells to recover from the electric pulse and to allow them to express the antibiotic resistance. After recovery, plate aliquots on selective media and incubate under normal conditions
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