A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Clark, J. A look at transformation efficiencies in E. Google Scholar. Curtiss, R. III, Inoue, M.
Scott and R. Dagert, M. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6, 23— Day, M. Miller and M. Dower, W. High efficiency transformation of E.
Nucleic Acids Res. Dreiseikelmann, B. Translocation of DNA across bacterial membranes. PubMed Abstract Google Scholar. Enea, V. Genetic studies with heteroduplex DNA of bacteriophage f1. Asymmetric segregation, base correction and implications for the mechanism of genetic recombination. Gigova, L. Cloning of nifM-like gene from Sinorhizobium meliloti. Comptes rendus de l'Acad'emie bulgare des Sci.
Hanahan, D. Studies on transformation of Escherichia coli with plasmids. Klebe, R. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25, — Kushner, S. Amsterdam: Elsevier. Lacks, S. London: Chapman and Hall.
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Meselson, M. DNA restriction enzyme from E. Nature , — Nikaido, H. Molecular basis of bacterial outer membrane permeability. Norgard, M. Gene 3, — Panja, S. How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli? Divalent cations neutralize these negative charges thereby reducing the repulsion Figure 5. Divalent cations promote intramolecular static attractive forces in the extracellular DNA. This induces the vector DNA to adapt a compact ball-like geometry which readily shuttles in through the membrane pores of the recipient cells.
How does temperature shock aid in bacterial transformation? Prolonged incubation on ice Figure 3 stiffens the lipid structures in the recipient cell membranes resulting in loss of their fluidity.
The high temperature 42 0 C heat shock is thought to increase the Brownian motion in the extracellular environment. Just to clarify, Brownian motion is random particle movement within fluid.
This helps pushthe external vector molecules to the interior of the cells. The transition from 0 0 C to 42 0 C leads to lipid loss in the cell membrane, while the subsequent drop to 0 0 C again causes protein loss. So, the application of cold-heat-cold shock is critical in this procedure to facilitate creation of permeating pores by depleting the cell membranes of both proteins and lipids. Further, in this process, depolarization of the membrane occurs, reducing the repulsion between similarly charged vector DNA and host cell membranes.
Stabilizing freshly transformed cells Both methods of transformation cause significant stress on the cells. Phenotypic lag - the time between recovery and expression As we discuss this idea of recovery and stabilizing your competent cells, we need to introduce you to the concept of phenotypic lag.
Figure 6. Schematic illustration of phenotypic lag. Recovering transformed bacterial colonies As shown in figure 6, after accounting for the phenotypic lag, the bacteria are plated on selective solid media. In case the transformation efficiency is high, plating the whole of the 1ml culture onto a single plate might produce a lawn growth without discernible isolated colonies. Sometimes two or more colonies stick to each other giving a false impression of a big single colony.
Obtaining single isolated colonies is a necessity in this procedure. As you will see in the next step, not all transformants have the right construct despite having the vector backbone which confers the antibiotic resistance phenotype.
Further, due to antibiotic degradation in localized areas of the plate, small satellite colonies emerge which are antibiotic-sensitive vector-negative bacterial populations. Streaking out a second time gets rid of these as well. Further screening of transformants to recover the desired colonies While the previous step selects in the transformed cells by eliminating the non-transformed cells by using selection pressure antibiotic , further screening of the transformants is indeed required to identify the bacterial colonies that have the desired recombinant vector construct.
Why do we need to screen transformant colonies? If the ligation mix is directly used as a source of the recombinant vector DNA in the transformation step, it would have all these DNA molecules in varying proportions: insert fragment linearized vector self-circularized by autoligation vector correct recombinant vector with the insert ligated within it at the correct position and sequence.
Cells with blank no insert self-recircularized vector 3 would produce colonies on the antibiotic plate. Cells with recombinant vector having the cloned transgene 4 would also produce colonies on the antibiotic plate. Transformants with possible variants of the desired recombinant vector consider a multi-fragment cloning experiment where only some but not all insert fragments have been cloned in the vector will also produce colonies.
Experimental tip: The colony is picked up from the agar plate using a sterile toothpick or pipette tip and is first patched onto a second plate before being added into the corresponding PCR tube Figure This serves the following two purposes: Too much bacterial cells in the PCR mix often is detrimental to the reaction, producing smudged unclear bands.
Patching onto a second plate reduces the cell load going into the PCR tube. For ensuring aseptic microbiology practice, a colony where a pipette tip or toothpick albeit autoclaved has touched should not be again used to start a culture in case it indeed is confirmed by PCR to be a desired strain and store it.
The second plate which now has the replica of this colony can be used for this purpose. When choosing the primer pair, one or many of the following 3 options can be chosen Figure 11 : Primers that bind at the two ends of the insert fragment Insert-specific. The colony that has the insert integrated within the plasmid produces a band on the DNA agarose gel, while the ones with only the blank vector produces no band.
However, in this case it would be difficult to differentiate between the cases of a true negative colony blank plasmid versus the PCR not working for a colony for any reason. The above problem can be overcome if primers bind to the plasmid backbone just upstream and downstream of the insert cloning site backbone-specific. The colonies with empty vectors produce shorter bands compared to those with the insert. Primers may be designed to ensure that the insert has been integrated within the vector in a specific orientation orientation-specific.
This is especially helpful in cases where the insert-vector cloning lacks directionality for example, blunt-end cloning or when only one RE is used to digest the vector..
Blue-white screening Blue-white screening is a method where positive colonies containing the insert in the plasmid construct can be readily identified visually and contrasted from the colonies transformed with empty vectors. Both this plasmid and the insert are digested with the same pair of restriction enzymes RE1 and RE2 , thus producing corresponding complementary overhangs depicted by sky-blue and violet colors b The digested plasmid fragments and insert are there in the ligation reaction.
Here two outcomes are possible: i the plasmid may self-circularize using the digested cut out plasmid fragment containing the lacZ gene which has sticky overhangs complementary to the plasmid backbone ii the insert may ligate with the plasmid backbone owing to its complimentary sticky ends, thus creating the recombinant vector c Competent bacteria are transformed directly with the ligation mixture which has both plasmids i and ii.
However, only the one with the self-circularized plasmid i will produce blue colonies due to expression of the betagalactosidase enzyme which cleaves X-gal to produce blue color metabolite due to the presence of the functional lacZ gene; the second type of transformant colonies will produce regular white colonies because the recombinant plasmid ii they harbor does not have the lacZ gene Colony PCR vs Diagnostic Digest vs Blue-white Screening Time consumption: The blue-white screening method allows ready visual differentiation between negative and positive colonies.
The diagnostic digest takes the maximum time because the additional steps of growing colonies as liquid cultures and isolating plasmids from them are involved. Cost: The diagnostic digest is most costly, due to the involvement of multiple restriction endonuclease enzymes.
IPTG , the chemical used to switch on the lac operon, and X-gal are not comparably expensive. However , blue-white screening is unable to confirm the orientation of the insert, which the other two methods can, if the experiment is properly designed Figures 11 and Further, in case multiple insert fragments need to be cloned in tandem consider Gibson assembly , the blue-white method cannot confirm whether all or only a subset of the required fragments has been cloned in a colony.
Bacterial transformation process workflow References Johnston. C et al, Bacterial transformation: distribution, shared mechanisms and divergent control.
Nature reviews microbiology, 12, Griffith F. The Significance of Pneumococcal Types. OT et al, Studies on the chemical nature of the substance inducing transformation of pneumococcal types.
A et al, Revisiting the mechanisms involved in calcium chloride induced bacterial transformation. Frontiers in Microbiology. Green MR, Sambrook J. Cloning and transformation with plasmid vectors. In: Molecular Cloning: A laboratory manual 4th ed. Cold Spring Harbor Laboratory Press. Studies on transformation of Escherichia coli with plasmids. J Mol Biol 4 Note to self - format in commercio to bring attention.
Tags bacterial transformation Pallabi Roy Chakravarty. Related Articles. Bacterial transformation is a genetic process where bacterial cells take up foreign DNA from the ext Molecular Cloning: A Detailed Introduction. Molecular cloning is a primary procedure in contemporary biosciences. This involves introducing a sp What is Agrobacterium? Agrobacterium is a naturally occurring soil bacterium with the unique ability How to choose the right Agrobacterium vector for plant transformation.
One of the key factors influencing an Agrobacterium-mediated gene transformation is the vector sel This gives them an evolutionary advantage and helps them survive changes in their environment.
For example, bacteria can acquire DNA that makes them resistant to antibiotics. The bacterial genome is contained on a single, circular chromosome. This genetic material floats freely in the cell , unlike eukaryotic organisms where the genetic material is enclosed within a nuclear membrane. Bacteria may sometimes contain smaller circles of DNA, called plasmids, which have a much smaller number of genes.
Plasmids can be swapped between bacteria in a process called conjugation. Plasmids can be used as vectors to carry foreign DNA into a cell. In the lab, plasmids are specifically designed so that the DNA they contain will be copied by bacteria. Laboratory-designed plasmids contain a small number of genes that help transformation. These include:.
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