大旨提醒：Transformation of Gram-Positive Bacteriaan adaptation from Chang, D., Chassy, B., Saunders, J., Sowers, A. 壹玖玖伍.Guide
骨干提醒：Introduction Quality is important in all aspects of tissue culture since the quality of materials used i.e. media and
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 bacteriaStart 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 CellsThe 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 and time constant . Typically, cocci will require higher field strengths 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 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 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 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 .
Quality is important in all aspects of tissue culture since the quality of materials used i.e. media and other reagents) will affect the quality of the cultures and products derived from them. The main areas of quality control that are of concern for tissue culture are:
- The quality of the reagents and materials
- The provenance and integrity of the cell lines
- The avoidance of microbial contamination
Reagents and Materials
A potential source of contamination is reagents and materials, in particular bovine serum which has been identified as a source of bovine viral diarrhoea virus . Porcine trypsin is also a potential source of Mycoplasma hyorhinis. Good quality reagents and materials are available from numerous manufacturers of tissue culture media and supplements. In addition manufacturers including Sigma will carry out a range of quality control tests including screening for mycoplasma and BVDV and supply a Certificate of Analysis with their products. These state the product and lot numbers and forms a vital part of record keeping and tracking of reagents used in the production of cell stocks. It is advisable to further test key reagents such as FBS to ensure that they are ‘fit for purpose’ due to batch-to-batch variation.
Manufacturers of sterile plastic ware designed for tissue culture use are also supplied with Certificates of Analysis for each batch produced, which should be kept for future reference.
Provenance and Integrity of Cell Lines
The sourcing of cell lines can have an important effect on quality since freshly imported cell lines are a major source of contamination. The advantages of obtaining cell lines from a recognized source such as a culture collection are:
- Contaminant free
- Fully characterized and authenticated in terms of DNA profile and species of origin
- Supplied with a detailed data sheet
Once cell lines have been obtained from a reputable source it is important to implement master and working cell banking procedures and the associated quality control steps such as routine testing for microbial contaminants and confirming the identity of cultures.
Avoidance of Microbial Contamination
Potential sources of contamination include other cell lines, laboratory conditions and staff poorly trained in core areas such as aseptic techniques and good laboratory practice. Thus the use of cells and reagents of known origin and quality alone is not sufficient to guarantee quality of product ; it is necessary to demonstrate quality throughout the production process and also in the final product. Routine screening aids the early detection of contamination since all manipulations are a potential source of contamination.
The 3 main types of microbial contaminants in tissue culture are:
- Bacteria and Fungi
Bacterial and Fungal ContaminationBacterial contamination is generally visible to the naked eye and detected by a sudden increase in turbidity and color change of the culture medium as the result of a change in pH. The cell culture may survive for a short time but the cells will eventually die. Daily microscopic observation of cultures will ensure early detection of contamination and enable appropriate action to be taken as soon as the first signs of contamination become apparent . In addition specific tests for the detection of bacteria and fungi should be used as part of a routine and regular quality control screening procedure .
Mycoplasma ContaminationMycoplasmas are the smallest free-living self-replicating prokaryotes. They lack a cell wall and lack the ability to synthesize one. They are 0.35m in diameter and can be observed as filamentous or coccal forms. There are 5 major species that are tissue culture contaminants, namely M. hyorhinis, M. arginini, M. orale, M. fermentans and Acholeplasma laidlawii.
The effects of mycoplasma infection are more insidious than those of bacteria and fungi inducing several long term effects. These include:
- Reduced growth rate
- Morphological changes
- Chromosome aberrations
- Alterations in amino acid and nucleic acid metabolism
However, despite these well-documented effects the presence of mycoplasma is often not tested for with the consequence that in such laboratories the majority of cell lines are positive for mycoplasma. Mycoplasma contamination is difficult to detect requiring the use of specialist techniques . In the past only specialist laboratories, such as culture collections, have performed these tests. However a variety of commercial kits are now available although the performance characteristics of these kits can be extremely variable. A combination of these should be used as part of a routine and regular quality control screening procedure. Culture collections such as ECACC are able to test cultures if required. Mycoplasma testing products are available from Sigma, refer to pages 489-490 of Life Sciences Catalogue.
Viral ContaminationSome cell lines contain endogenous viruses and secrete virus particles or express viral antigens on their surface . These cell lines are not considered contaminated. However, bovine serum is a potential source of bovine viral diarrhoea virus contamination. Use of infected serum will lead to contamination of cell lines with the virus. Contamination of cell lines with BVDV may cause slight changes in growth rate but since this virus is non-cytopathic macroscopic and microscopic changes in the culture will not be detected. Suppliers of bovine serum are aware of this and screen sera accordingly and generally serum is sold as BVDV tested.
It is good practice to monitor the laboratory environment where cell cultures and their products are prepared. Class II microbiology safety cabinets should be checked every 6 months to ensure that they are working efficiently. However it is also advisable to monitor the number of contaminants within the cabinet by periodically placing open settle plates on the cabinet work surfaces. In addition settle plates should be used to assess airborne microbial burden at selected points around the laboratory. Plates should be left open for a period of 4 hours. After this time they should be covered, placed in sealed boxes and incubated at 32ºC and 22ºC for up to 7 days. At the end of this period the plates should be examined for the presence of microbial growth. The position of each plate in the cabinet should be recorded and results stored for trend analysis.
Acceptable limits should be defined in terms of “alert” levels and “action” levels, the actual values being dependent on the criticality of the work and the levels of cleanness that can be achieved under normal operating conditions.
What to do in the event of contamination
One hugely under-estimated problem in tissue culture is the routine use of antibiotics. Continuous use of antibiotics is unnecessary and can lead to the development of resistant strains that are difficult to eradicate and may require the use of more exotic antibiotics that may be toxic to the cell cultures. In addition the use of antibiotics may mask a low level of contamination.
Once a contamination has been detected, whether is it due to bacteria, fungi or mycoplasma, the recommended course of action is to discard the culture and continue the work with earlier stocks that are known to be free of contaminants or obtain fresh stocks from a recognized source. However if this is not possible eradication of the contaminant may be attempted with the use of antibiotics. In addition culture collections such as ECACC will attempt to eradicate any contaminants if required. Please contact ECACC for further details.
Viral infections are virtually impossible to remove from cultures since they do not respond to antibiotic treatment. Also, since they are intra cellular parasites it is not possible to remove them by centrifugation or other separation techniques. If virus free stocks or a virus free alternative is not available then a thorough risk assessment should be undertaken prior to continuing work with the infected cell line.