When browsing competent cells for your research, you might encounter some unfamiliar names of bacterial strains, terminology, and features. To help you choose your competent cells, this article provides you with information about some considerations, comparisons, and strains.
In this article:
How Do I Choose the Right Competent Cells for My Experiment?
One important consideration before choosing competent cells is whether you are planning to perform heat-shock transformation or electroporation.
You can get higher transformation efficiency by performing electroporation than heat-shock transformation. But you must remove salts, such as calcium chloride, from your competent cells to avoid an electric arcing. Therefore, for electroporation, only use electrocompetent cells made with salt-free approaches.
Electroporation also requires an electroporator. If your laboratory is not equipped with an electroporator, choose chemically competent cells for heat-shock transformation.
Transformation efficiency of competent cells indicates cells containing the cloned DNA after transformation. The higher the number is, the more efficient the competent cells are. Therefore, this number is an important factor to obtain your desired clones.
Generally, competent cells with adequate transformation efficiencies (106- 108 cfu/µg) are suitable for cloning and subcloning many regular size plasmids (<10 kb).
The efficiency of your competent cells matters more when performing a challenging experiment, such as cloning large plasmids. When using less efficient competent cells, you can get fewer positive clones, or worse, none. Therefore, if you’ve found your ligations to be incredibly difficult, try GoldBio competent cells with ultra-high transformation efficiency (>1010 cfu/µg), for example GB10B-Pro™.
3.Genotype of the Cells
Understanding genotype and properties of the cells helps you to achieve anticipated results after transformation.
For example, some bacterial strains have a partial deletion of the lacZ gene in the alpha region, represented as lacZΔM15. The strains with this genotype allow you to perform blue and white screening after transformation and pick your transformed cells.
To learn more about the different genotypes of Escherichia coli, read this GoldBios article:
Before selecting competent cells, you need to consider the expected results of your transformation. For protein expression, you will need a different strain of competent cells than what you would use for regular cloning.
Through the rest of this article, we’ll discuss different downstream applications, and which types of competent cells are most appropriate for those methods.
What Competent Cells Should I Use for Cloning and Subcloning?
For cloning and subcloning, choose competent cells with ultra-high transformation efficiency, such as GoldBio GB5-alpha™ (electrocompetent cells or chemical competent cells) or GB10B™ alpha (electrocompetent cells or chemical competent cells).
The GB5-alpha™ has the recA1 mutation, preferable for maintaining cloned DNA stability. In addition, it has the endA mutation to prevent cleavage and degradation of DNA, resulting in high quantity and quality of DNA. This strain is efficient for cloning unmethylated DNA from PCR or cDNA.
GB10B™ has recA1 and endA1 mutations, resulting in high quality DNA. It also carries mutations for mcrA, mcrBC, and mrr. McrA, McrBC and Mrr are enzymes encoded by mcrA, mcrBC, and mrr. These enzymes are part of restriction-modification systems in which digestion of methylated DNA occurs. Therefore, the strain with these gene mutations allows cloning of methylated DNA, particularly for cloning eukaryotic genomic DNA.
What are Good Competent Cells to Use for Transformation of Large Plasmids?
GoldBio’s GB10B-Pro™ competent cells are suitable for transformation of large plasmids, ranging from ≥10 kb up to 350 kb. Similar to GB10B™, GB10B-Pro™ can also be used for blue- white screening protocol and it supports DNA methylated cloning. In addition, this strain can produce a high number of transformed cells for more difficult cloning.
What are Competent Cells to Use for Genomic and cDNA Library Construction?
If you are planning to build genomic and cDNA libraries, consider E. coli GB10B™ electrocompetent cells or chemical competent cells) or GB10B-Pro™ electrocompetent cells. Both strains have ultra-high transformation efficiencies and allow cloning of methylated DNA.
Another option is to use competent cells from another bacterium, for example Lactococcus lactis. Both MG1363 and IL1403 can also be used for genomic and cDNA library construction.
What are Competent Cells to Use for Protein Expression?
For recombinant protein expression, you can use BL21 or BL21 (DE3) (chemically competent cells and electrocompetent cells). BL21 and BL21 (DE3) belong to B strain, deficient in Lon and OmpT proteases, therefore reducing the degradation and promoting the stability of your synthesized proteins. The addition of IPTG into the growth medium induces the production of proteins for both strains.
To understand how IPTG induces protein synthesis, read this GoldBio article:
The BL21 strain is appropriate for transformation and routine protein expression from a vector with no T7 promoter. The T7 promoter is a sequence of DNA, recognized by T7 RNA polymerase.
BL21(DE3) carries the DE3 phage encoding for T7 RNA Polymerase. This strain is suitable for protein expression with the T7 promoter. You can also select this strain for use with other promoters recognized by the E. coli T7 RNA polymerase, for example: lac, tac, trc, ParaBAD, PrhaBAD, and T5 promoter.
What is a Good Competent Cell for Phage Display?
For phage library screening, choose TG1 Phage Display. TG1 Phage Display, an amber suppressor (supE) strain, suppresses the amber nonsense mutation (mutation that inserts UAG stop codons leading to truncated proteins). Therefore, this strain supports the phage growth and allows the incorporation of an amber stop codon between the displayed protein and the phage coat protein. This strain is highly efficient for phage display.
What are Competent Cells to Use for Plant Transformation?
The Agrobacterium AGL1 carries the hypervirulent and weakened tumor-inducing plasmid pTiBo542, allowing optimal DNA transformation. The AGL1 strain has a mutation in its recA gene, supporting the stability of cloned DNA. This strain also has carbenicillin resistance marker for selection.
The Agrobacterium GV3101 carries the Ti plasmid pMP90 (pTiC58DT-DNA) with gentamicin resistance.
You can choose GV3101 for plant transformation of Arabidopsis thaliana, maize, and other monocots. Otherwise, use AGL1 for plant transformation of several dicots, including A. thaliana, tobacco, potato and soybeans.