loning strains are very specific bacterial strains used in molecular biology to clone recombinant DNA molecules. Characteristics of good cloning strains include being deficient in restriction enzymes, and having high transformation.
E. coli is the model lab organism for gene cloning. Specifically, GB5-α™ and GB10B™ are E. coli strains optimized as excellent cloning host cells.
They grow well, have high transformation efficiency to maintain plasmid numbers and fidelity, and can be used for blue-white screening to monitor cloning results.
In this article, we will take a closer look at what cloning strains are, and examine the characteristics of the two most common cloning strains used in labs.
What are cloning strains?
Cloning strains, also known as host cells, are organisms or cell lines used in cloning techniques to replicate and produce copies of specific DNA sequences with high fidelity.
Cloning strains are chosen for their ability to efficiently incorporate foreign DNA, replicate it, and maintain the stability of the cloned DNA during propagation. These strains serve as a foundation for cloning experiments and are essential for the production of recombinant proteins and other genetic manipulations.
They are different from expression strains, which are optimized for expressing the cloned recombinant DNA.
A strain that serves as a good host cell for cloning should have the following characteristics:
- Fast growth on solid and liquid media
- High transformation efficiency
- Maintains and replicates the vector along with the cloned transgene without incurring mutations in them.
- Enables visual differentiation between desired and undesired clones.
- Sensitive to most antibiotics, so selectable markers in cloning vectors can be utilized for screening.
- Absence of enzymes called endonucleases that break down vector molecules such as plasmids.
E. coli, the most common cloning organism used in the lab is naturally sensitive to most antibiotics. Antibiotic resistance requires specific genes to be present in the organism that confers the resistance phenotype. E. coli naturally lacks these genes.
Along the same lines, E. coli naturally is a fast grower in culture, with a doubling time of only 20 minutes.
As for the other features, such as a lack of enzymes to chop off or mutate vectors and the cloned DNA – these have been specifically modified by scientists to make these strains optimized for cloning. We will look at these genetic features further in this article.
In addition to having features helpful for gene cloning, both GB5-α™ and GB10B™ are considered safe to handle. They lack virulence factors that may cause disease in the person handling them.
GB5-α™
E. coli strain GB5-α™ is widely used as a host strain for gene cloning. Mutations in recA1 and endA1 ensure that the introduced plasmid does not recombine with the host genome and is not cleaved by host endonucleases, respectively.
It also features α-complementation for blue-white screening.
In this section we will look at the prominent genetic characteristics that GB5-α™ has, which are very helpful during gene cloning. We will start with the recA1 mutation.
recA1
RecA facilitates homologous recombination. When transforming a cloning host strain with a plasmid, you would not want recombination between it and the host genome – to maintain sequence fidelity of the vector and the transgene cloned in it. The recA1 mutation in GB5-α™ ensures this.
To understand why homologous recombination should be turned off in cloning strains, please refer to this article on homologous recombination.
As shown in figure 2, if a strain expresses RecA, homologous recombination might occur between an introduced plasmid and the host cell’s genome.
During cloning, you would want the sequence of the vector and the transgene to remain exactly as it is, after you introduce them in the cloning host, which is why the recA1 mutation is so important.
endA1
Endonuclease 1 cleaves double-stranded DNA. Vectors and the transgenes cloned in them are double-stranded and can potentially be cut up by Endonuclease 1 if expressed by the cloning host.
GB5-α™ does not produce Endonuclease 1 due to the mutation in endA1.
This ensures that the vector and the insert are preserved properly inside the cell and are not cleaved and degraded.
α-complementation
α-complementation is a phenomenon where a mutation in the lacZ gene (lacZΔM15) is present in the cloning strain GB5-α™, along with a corresponding genetic element in the cloning vector used during transformation. This combination enables blue-white screening of colonies to figure out which ones have the transgene cloned in them.
GB5-α™ has a mutation - lacZΔM15. Because of this, it expresses a β-galactosidase protein with a large portion missing in the amino terminus. This mutated β-galactosidase protein is called ω-peptide.
For the β-galactosidase protein to function as an enzyme, the full N-terminal is required which helps the protein form a tetramer required for its catalytic activity. Due to the deletion of critical amino acids in the N-terminal, the ω-peptide cannot tetramerize.
So, GB5-α™, due to the lacZΔM15 mutation, can express only the non-functional ω-peptide.
When untransformed GB5-α™, that is – without the corresponding cloning vector, is plated on media containing X-gal, it forms only white colonies.
Figure 1. Schematic showing ω-peptide alone cannot form functional β-galactosidase protein resulting in only white colonies.
For gene cloning, GB5-α™ is used in combination with a suitable cloning vector, for example pUC-19. The multiple cloning site of this plasmid has the gene lacZα, that encodes for the first 59 amino acids of the N-terminus of β-galactosidase protein called LacZα.
Now, when you transform GB5-α™ with this suitable corresponding plasmid, the ω-peptide expressed by the host genome and the LacZα come together to form the full β-galactosidase protein that can now tetramerize and show enzymatic activity. This is called α-complementation.
So, when GB5-α™ is transformed with a plasmid such as pUC-19 that has lacZ-α in its multiple cloning site, the colonies are all blue on a plate containing X-gal (figure 2).
Figure 2. Schematic showing alpha-complementation resulting in blue colonies.
When a transgene is cloned into the multiple cloning site of this plasmid, it disrupts the lacZα gene. So, when GB5-α™ is transformed with this plasmid that has a transgene cloned into its multiple cloning site, there will be no α-complementation. The desired colonies will be white when plated on media containing X-gal.
Figure 3. Schematic showing how a transgene cloned in the multiple cloning site of plasmid disrupts lacZα gene with no α-complementation, resulting in only white colonies.
You might wonder how would it be possible to differentiate between non-transformed GB5-α™ cells from those GB5-α™ cells that have the plasmid with the insert cloned into it– because both would produce white colonies on a plate containing X-gal.
Well, this would not be a problem at all because the plasmid contains an antibiotic selectable marker, and your media would have the corresponding antibiotic as the selection pressure.
So, non-transformed GB5-α™ would not grow in this plate because it would be sensitive to this antibiotic. Only those GB5-α™ cells that were transformed would grow.
And then, among the surviving cells, those with the plasmid + insert, would grow as white colonies. The ones with the blank plasmid would produce blue colonies.
So, in this experiment you are using selective and differential media at the same time.
What we’ve discussed so far covers the theoretical details. It is a bit more complicated when designing the actual experiment.
For example, to get GB5-α™ cells to actually start expressing the ω-peptide, you would need to induce with IPTG. You can find more information about that in this article.
You can perhaps now appreciate how versatile and useful GB5-α™ is as a cloning host.
It ensures that the plasmid (along with the insert, if applicable) is replicated without mutations, is not cleaved and degraded by non-specific host endonucleases, and it also lets you do blue-white screening.
Nevertheless, GB5-α™ has a few shortcomings that might hinder cloning in some cases – like if you are cloning genes of eukaryotic organisms or using very large vectors such as Bacterial Artificial Chromosomes (BACs).
To tackle such situations, scientists use another handy cloning strain, GB10B™ .
GB10B™
Like GB5-α™, GB10B™ has recA1, endA1 and lacZΔM15 mutations. Additionally, GB10B™ has enzymes deleted that cleave methylated DNA, enabling you to clone eukaryotic DNA sequences that are often methylated. Also, GB10B™ has constitutive deoxyribose synthesis that helps in cloning procedures that involve large vectors.
In comparison to GB5-α™ , GB10B™ is more suitable for cloning eukaryotic DNA and with large vectors. We will discuss how.
MDRS gene loci (mrr, mcrA, mcrB, and mcrC) deletion
The MDRS gene loci deletion is a feature in GB10B™ that is helpful in cloning eukaryotic genes.
These genes encode enzymes that cleave methylated DNA, which is common in eukaryotic genomes. Because of this mutation, methylated DNA can be cloned using GB10B™ as the host.
Enzymes encoded by the MDRS loci are kind of an immune response in regular E. coli. Whenever DNA from other organisms enter E. coli cells, they can recognize it as foreign because these foreign DNA are often methylated. And then these enzymes cleave those foreign DNA molecules.
Now, this can be a huge problem in case you are cloning a methylated DNA fragment using a regular E. coli strain as the cloning host organism. Your cloned insert will get destroyed once introduced into this host.
There lies the importance of the MDRS mutation in GB10B™.
Cloning using large vectors possible using GB10B™
GB10B™ is a handy cloning strain especially when you are using a large plasmid or another large vector such as BAC. The generally accepted reason behind this is that GB10B™ has constitutive deoxyribose synthesis.
The correlation between deoxyribose synthesis and higher efficiency for cloning with large vectors is not clear. Perhaps, more deoxyribose ensures seamless replication of the large vectors – the sugar (deoxyribose) required in the large sugar-phosphate backbone would not be a limitation.
The mutation in the gene deoR was initially thought to be the reason for constitutive deoxyribose synthesis in GB10B™. However, a later study reported that 10B is in fact not a mutant in deoR. So, why GB10B™ is efficient in cloning with large vectors is not very clear yet.
So far, we saw why GB5-α™ and GB10B™ are so commonly used as cloning strains. While these two strains rank very high in terms of how frequently they are used as cloning host organisms, please keep in mind that there are several other cloning strains – many are even from the same E. coli background.
One issue that both GB5-α™ and GB10B™ have is that they have methylase enzymes that methylates adenine and cytosine residues by a site-specific manner.
The problem is that if you introduce a plasmid, with or without an insert gene fragment, in these strains, the DNA there might get methylated. This might mess up the next steps in the cloning process.
For instance, if you want to isolate the plasmid from these methylase+ strains and digest them with restriction endonucleases (REs), you might be unsuccessful because methylation at restriction endonuclease recognition sites interfere with digestion for multiple restriction endonucleases; for example, MboI.
Replication of plasmids isolated from methylase+ strains, in methylase- strains, may also be problematic.
In such cases, plasmids are propagated in strains such as JM110, which are devoid of methylases.
The other consideration you might want to have is what you would want to do downstream of your cloning process.
If it is mass producing the product of the cloned transgene, you might want to subclone into a cloning vector in a cloning strain.
If the cloning is intended for plant biotechnology purposes, then perhaps you’d have to subclone into Agrobacterium tumefaciens.
For a detailed guide on choosing your appropriate cloning host, depending on what you want to do finally with your cloning, please refer to this article.
All in all, GB5-α™ and GB10B™ are highly valuable cloning strains in molecular biology. GB5-α™ offers recombination and endonuclease protection, along with α-complementation for blue-white screening. GB10B™ excels in cloning methylated DNA and large vectors. These strains empower researchers to achieve precise gene manipulation and drive advancements in genetic research.
E. coli Strain Comparison Chart
Table 1. E. coli Cloning Strains Comparison Chart
References
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Grant S et al. 1990. Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. 87(12): 4645-9
Hanahan D. 1991. Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 204: 63-113
Marinus and Morris. 1973. Isolation of deoxyribonucleic acid methylase mutants of Escherichia coli K-12. J Bacteriol. 114(3):1143-50
May and Hattman. 1975. Analysis of bacteriophage deoxyribonucleic acid sequences methylated by host- and R-factor-controlled enzymes. J Bacteriol. 23(2):768-70
Moosmann and Rusconi. 1996. Alpha complementation of LacZ in mammalian cells. Vol 24(6): 1171-1172
Russell and Zinder. 1987. Hemimethylation prevents DNA replication in E. coli. Cell. 50(7):1071-9
