Agrobacterium 22 , Burkholderia 13 , Photorhabdus 23 and Xenorhabdus It was inconvenient to maintain low temperature conditions for preparation, storage and transport of the electrocompetent cells.
We intended to test the transformation efficiency of the electrocompetent cells prepared at room temperature. A large plasmid pGB-amp-Ptet-plu The warm electrocompetent cells showed 10 times higher transformation efficiency than the cold electrocompetent cells Fig.
Cells were transformed by 0. Plasmids were varied in the size, selection marker and origins of replication. Initially we tested three plasmids with different sizes. Two of them were p15A origin plasmids with ampicillin amp or chloramphenicol cm resistance. Another one was a pBR origin based plasmid with ampicillin resistance.
All the plasmids gained higher transformation efficiency with room temperature electrocompetent cells Fig. We also tested BAC vectors with different size and selection markers. All BACs gained higher transformation efficiency when room temperature electrocompetent E. These results indicated that for electrocompetent transformation, room temperature electrocompetent cells were more efficient than cold electrocompetent cells irrespective of their size, selection marker and origins of replication.
Therefore the room temperature electrocompetent cells could be a better candidate for gene cloning, construction of DNA libraries and mutagenesis than cold electrocompetent ones. The E. Along with this strain, several other commonly used E. Results revealed that although different E. We also tested the improving approach in a few of other Gram-negative bacterial strains.
An oriV origin plasmid pRK 2 -apra-kan based on plasmid pBC 27 , 28 , was utilized for transformation. When PG1 competent cells were prepared at room temperature, the electroporation efficiency of RK 2 plasmid was around three times higher than the cells prepared on ice Fig.
Other Gram-negative bacterial strains, such as Agrobacterium 22 , Burkholderia 13 , Photorhabdus 23 and Xenorhabdus 23 , were tested for RK 2 plasmid transformation by using room temperature and cold temperature protocols.
All the results indicated that room temperature competent cells had higher transformation efficiency than cold competent cells Fig. The plasmid transformation efficiency significantly increased by room temperature electrocompetent cells was not the destination.
It was necessary to evaluate the improvement of the room temperature protocol on lambda Red or Rac RecET mediated recombineering. The results showed that LLHR in room temperature competent cells was 6—10 times more efficient than the cells prepared on ice.
Both p15A origin and pBR origin plasmids gained the same fold increase Fig. PCR cloning is a routine exercise in every molecular biology laboratory It is thus our interest to find out an easy and inexpensive way to clone PCR products.
Since the electrocompetent cells prepared at room temperature improves the LLHR efficiency around 10 folds, it is essential to find out the minimum homology sequences needed for LLHR.
Normally, after 2. When bacterial cells were overgrown, the transformation efficiency dropped down Fig. It was noteworthy that over-grown or even overnight cultured bacterial cells could still be used for transformation when room temperature protocol was used for preparing competent cells. To predigest the transformation process, we had tested whether the recovery step could be omitted. For simple plasmid transformation, the recovery step could be omitted when the electrocompetent cells were prepared by using room temperature protocol Fig.
Results suggested that plasmid or ligation transformation could be performed in a few minutes after electroporation by using room temperature competent cells. Previous results concluded that the room temperature electrocompetent cells had much better transformation efficiency than cold electrocompetent cells. Furthermore we wanted to know how long the competent cells could stay at room temperature without any significant loss of transformation efficiency.
These results indicated that the room temperature competent cells lost their transformation efficiency to the maximum when stored in room temperature more than one day. This ability gives us an opportunity in the future to deliver the competent cells in routine cooling pack, which is easier and cost effective.
To find the reasons of higher efficiency in room temperature protocol, electron microscopy was used for comparative analysis of the morphological shapes of cold competent cells and room temperature competent cells of E. Their comparative analysis showed that cold competent cells appeared to shrink more than room temperature cells, and the surface of room temperature competent cells was found smoother Fig 3a—d. Shrunken cells might be more difficult to transform, and the bacterial cell membrane and wall could be more permeable for foreign DNA entry at a higher temperature.
Additionally, it may be difficult for the shrunken cells to form pores that allow DNA transfer through the cell membrane under electroporation conditions, and after electroporation most of the cold competent cells were found to be lysed. The ability to introduce exogenous DNA molecules into the cells plays key role in the development of molecular biology techniques, such as mutagenesis and genetic engineering of microorganisms. Several methods have been reported to introduce exogenous DNA molecules into the cells which includes chemical treatment, electroporation, utilization of a biolistic gun, polyethylene glycol, ultrasound, microwave, and hydrogel In those methods, electroporation has been often demonstrated to be more efficient and convenient way to transform a large number of microorganisms used for genetic studies 32 , and many efforts have been performed to increase its efficiency 33 , 34 , 35 , The phage-derived homologous recombination systems have been developed into very useful DNA engineering technologies, well known as recombineering which has also been performed in electrocompetent cells 10 , This suggests that a crucial step in recombineering is the transformation of E.
In conventional electroporation transformation, the electrocompetent cells were prepared on ice and the other supplies were also in cold environment, including pre-chilled cuvette, buffer, and centrifuge. The cells must be repeatedly washed before electroporation to remove conductive solutes. If the conductivity of a cell mixture is too high, then arcing will occur during electroporation, which will ruin the experiment.
The washing process can elicit a stress response that can lead to decrease in transformation efficiency. However, current studies reveals that the electroporation transformation efficiency is decreased at ice-cold temperature Fig.
This decreased efficiency might be due to ice cold temperature which alters the cell membrane topology. The cell membrane mainly consists of phospholipids and proteins 37 and the phospholipid bilayer forms a stable barrier between two aqueous compartments.
Embedded proteins of phospholipid bilayer carry out the specific functions of the plasma membrane, including the selective transportation of molecules across the membrane and cell-cell recognition At ice-cold temperature, the fatty acid tails of the phospholipids become more rigid. Therefore, cold temperatures may be not favourable for the survival and thus decreases the transformation efficiency.
Previous reports revealed that if the environmental conditions were changed, including the temperature, the cell membranes undergoes a gross morphological changes These structural perturbations were associated with characteristic disturbances of functions such as loss of selective permeability. Similar results were observed in this study that cold competent cells were appeared to shrink more than room temperature cells, and additionally more cold competent cells were found lysed after electroporation Fig.
The temperature effects on electroporation transformation could be explained by thermal effects during electro pore formation 41 , 42 , According to the electroporation theory, hydrophobic pores in the cell membrane were formed spontaneously by lateral thermal fluctuations of the lipid molecules 39 , which suggested that hydrophobic pores formation would be enhanced by increased temperature conditions.
To improve recombination efficiency many parameters had been described previously 17 , 44 , 45 except the transformation efficiency.
This study showed that LLHR efficiency in room temperature competent cells was higher than in the same cells prepared on ice Fig. The Red recombinases Red alpha and beta might be not stable while preparing the competent cells at room temperature. In conclusion, this study reports an unexpected finding, that is contrary to common assumption, that it is better to prepare bacterial cells at room temperature than on ice for electroporation. In addition, this study also shows that this is not only efficient for E.
However, further research will be essential to confirm the transfer and principle of membrane in competent cells. The bacterial strains and plasmids used in this study were listed in Table S1. Cells that undergo reversible electroporation can survive the effects of membrane permeation and recover the cell membrane can reseal afterward. Molecular biologists use this technique to temporarily permeate the cell membrane in order to introduce foreign molecular material, such as DNA, into a cell.
Unlike reversible electroporation where membrane nanopores are temporary, irreversible electroporation creates permanent pores that ultimately leads to apoptosis programmed cell death. Researchers have been using IRE for tumor treatment. Therefore, IRE is not going to be a method used for molecular transformation and transfection. Blokesch, M. Natural competence for transformation. Current Biology , 26 21 , RR Chan, W. A comparison and optimization of methods and factors affecting the transformation of Escherichia coli.
Bioscience reports , 33 6 , e Chen, I. Journal of bacteriology , 10 , Cheng, C. DMSO induces dehydration near lipid membrane surfaces. Biophysical journal , 2 , Deipolyi, A. Irreversible electroporation: evolution of a laboratory technique in interventional oncology. Diagnostic and Interventional Radiology , 20 2 , Dybvig, K.
Artificial transformation of mollicutes via polyethylene glycol-and electroporation-mediated methods. Molecular and diagnostic procedures in mycoplasmology , 1 , Finkel, S. DNA as a nutrient: novel role for bacterial competence gene homologs. Journal of Bacteriology , 21 , Hanahan, D.
In Methods in enzymology Vol. Academic Press. Jain, R. Horizontal gene transfer among genomes: the complexity hypothesis. Proceedings of the National Academy of Sciences , 96 7 , LabBench Activity: Competent Cells.
Mell, J. Natural competence and the evolution of DNA uptake specificity. Journal of bacteriology , 8 , Napotnik, T. In vitro electroporation detection methods—An overview. Bioelectrochemistry , , The most common transformation methods are electroporation or heat shock transformation. The recovery step : the cells are incubated in a recovery medium to restore the cell membrane and the cell wall.
The advantages of using electroporation are the higher efficiency, more colonies, and much faster transformations compared to heat shock method. Transformation efficiencies for electroporation are 5. Therefore, electroporation is helpful when you have to construct DNA libraries. The drawback of this method is that you must have an electroporator, which is a special piece of equipment.
In addition, the common problem during electroporation is the presence of salts or air bubbles in your DNA, and in the cuvette, can cause an arcing. Unfortunately, this will make you lose your sample and require you to redo your ligation reaction. Heat shock transformation is relatively easy compared to electroporation. It is also simple, only requiring a water bath. You can use this method, when you only need to get a few positive clones.
To perform transformation, you must have competent cells. There are two types of artificially competent cells available: electrocompetent and chemically competent. What you use for electroporation is electrocompetent cells, whereas chemically competent cells are used for the heat-shock transformation method. Competent cells are bacterial cells commonly used for transformation. Transformation of bacteria involves the binding of foreign DNA to the cell membrane, and the movement of DNA across the membrane into the cytoplasm.
In electroporation, an electric pulse creates pores and a temporary electric field. The electric field pulls the DNA to the more positively charged end or into the cell. Preparing electrocompetent cells are relatively easier than making chemically competent cells. Glycerol, used for extensive washing, removes remaining salts from the pellet suspension. During the heat shock transformation, the heat pulse decreases the membrane potential of the competent cells, therefore lowering the potential barrier for the movement of negatively charged DNA into the cytoplasm Panja et al.
To make chemically competent cells, pellets are usually treated with salts, for example by using CaCl 2 or MgCl 2. An alternative option to making competent cells is using commercially-available strains. This eliminates many of the hassles associated with this time-consuming process and ensures optimal transformation efficiency, as it has already been measured and validated.
The best option for rapid and efficient transformation would be the Mix and Go! Competent Cells. Competent cells are one of the most commonly used reagents within the lab and having the right cells is crucial for any successful transformation. Whether you choose to purchase or make competent cells, the technical support team at Zymo Research is available help you through the process. Get news, product info, tips, industry updates, events, freebies, and more delivered right to your inbox.
Email Address Password Forgot Password.
0コメント