3 Things to Know for Making Competent Cells
Bacterial transformation is one of the most commonly performed techniques in molecular biology. This process transfers exogenous DNA (e.g. plasmids, gene deletion cassettes, etc.) into a host cell. Once within the cell, the DNA can then be incorporated into the genome, replicated, used to produce proteins, and more. However, transformation can be an inefficient process that requires methodological tricks to move the DNA past the bacterial membrane(s) and cell wall and make the bacteria “competent” for DNA uptake.
What is competency?
Many bacterial species can naturally uptake DNA from the environment. However, the most commonly genetically transformed lab bacteria, Escherichia coli, is not.
To overcome a lack of natural competence, E. coli can be treated by a number of procedures to render it able to take up DNA. Typically, researchers use chemical (and heat shock) or electroporation means to transform, although other methods exist. The process of making competent cells introduces pores into the cell membrane which allow they to uptake extracellular DNA more readily. Once these competency methods are complete, the E. coli cells are ready for DNA transformation.
What is transformation efficiency?
Transformation efficiency is commonly used to describe how well competent cells take up DNA. This value is described as the number of colony forming units (cfu) produced by transforming 1 µg of plasmid DNA for a given amount of competent cells. For example, an ideal efficiency would be 10 8 cfu/µg of DNA.
The transformation efficiency is affected by a variety of factors including the genotype of target cells, plasmid size, supercoiled vs. relaxed DNA, growth phase of cells at time of collection and method of transformation. Given the variety of potential confounding factors, special care must be taken to ensure successful transformation experiments.
Making Competent Cells
Most typically, competent cells are used in molecular cloning workflows, protein expression, and any a variety of applications using plasmid DNA. However, preparing the E. coli competent cells can be tedious, requiring extremely pure water, designated autoclaved glassware, and high-grade reagents, or even specialize equipment (electroporators), depending on the method of transformation. There are many protocols available that detail the process and buffers required to make competent cells. There are even kits available with pre-made buffers to speed up this process. Given these challenges, the list below can help you steer clear of problems when making competent cells.
To make chemically competent cells:
Keep them COLD!
The process of making competent cells is challenging due to the need for the cells to stay cold. This is crucial because the cells are so sensitive and fragile while they are being made competent. Keeping the temperature low helps to avoid cell death during processing.
This means using a cold room if possible and performing the centrifugations at 0 – 4 °C. Pre-cooled microcentrifuge tubes and racks should also be used and kept on dry ice before use. Doing this allows the cells to freeze faster, important especially when aliquoting the cells.
Keeping the cells cold is even important during the culturing process. Lower temperature growth (18 – 33 °C) increases transformation efficiencies. Due to this, we recommend using ZymoBroth, as it can promote efficient growth even at lower temperatures.
Prepare Freezer Space
Competent cells need to be stored at -80 °C. The process of making the cells competent makes them very fragile – likely to rupture and die. This means that storing at -20 °C can dramatically impede the transformation efficiency. After just 24 hours of storage at -20 °C, cells can lose up to 90% of the transformation efficiency.
Prepare reagents and labware prior to starting
Making competent cells can be a long and tedious process with several lengthy incubations. The process requires use of sterile growth media, glassware, and processing reagents. This requires preparation of all reagents and labware prior to starting the procedure.
The Easiest Way to Get Competent Cells
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. They have very high transformation efficiencies of up 109 transformants per µg of plasmid DNA and bypass the conventional heat shock procedure to perform transformations in 20 seconds (for ampicillin resistance-based plasmids).
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.