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. These include:. The piece of DNA or gene of interest is cut from its original DNA source using a restriction enzyme and then pasted into the plasmid by ligation.
The plasmid containing the foreign DNA is now ready to be inserted into bacteria. This process is called transformation. Before transformation, bacteria are treated with a chemical called calcium chloride, which causes water to enter into the cells and makes them swell.
These swollen bacteria are then known as competent bacteria. The plasmid DNA enter the bacteria through small pores created in the cell membranes. After transformation, bacteria are grown on a nutrient rich food called agar. The repulsion between foreign DNA and the bacterial cell, owing to negative charges on them both, are overcome by these divalent cations. It is thought that the divalent cations bind both to the cell and the DNA, thus neutralizing the charge altogether.
Moreover, DNA binding proteins present in the cell membrane could also aid in this interaction. Further, the low temperatures used in transformation protocols congeals the lipid moiety and consequently restricts the fluidity of the cell membrane which strengthens calcium-cell surface interaction. Clark et al.
The binding of calcium ions to the membrane also cause changes in the membrane permeability Li et al. Treatment with divalents or trivalents on ice is followed by treatment with elevated temperature as a heat-shock, which produces a temperature imbalance.
Molecules with increased Brownian motion outside the cell are likely to push the DNA molecule inside the cell. However, it is unclear if this kinetic force is sufficient enough to push the adsorbed DNA molecules inside.
Panja et al. It was inferred that lowering of temperature actually contributes to protein loss, while heating contributes to lipid loss, and thus together these cycles increase transformation efficiency Panja et al. Moreover, due to loss of lipids and proteins, the membrane is depolarized, further reducing the repulsion between the DNA molecule and the membrane. Moreover, cell density can also affect the efficiency of transformation and it has been reported that maximum competency is observed at cell density ranging from 10 7 to 10 8 cells ml in the log phase Taketo, ; Norgard et al.
However, the question remains; whether the pores through which foreign DNA enters a cell are formed by the calcium treatment or are they naturally present. There exist natural channels, often called Bayer's bridges, in the membrane that can serve as potential pathway for DNA uptake Dreiseikelmann, ; Sperandeo et al.
Hanahan stated that the competent cells have many sites or channels and all these sites and channels have an independent chance of taking part in the uptake of DNA moving toward the process of transformation. All the cells, whether competent or not, compete for the uptake of plasmid but if only competent cells are used for the transformation, the efficiency will be increased up to folds as discussed by Hanahan It was reported that the chances of transformation are not increased by increasing the concentration of DNA but by the increase in the number of channels through which the uptake of DNA takes place Hanahan, ; Nikaido and Vaara, Moreover, calcium has a dual role in this process; it not only neutralizes the charge but also weakens the cell membrane to produce invaginations Stein, ; Thomas and Rice, While it was known that the divalent cations help neutralize the charge, the complex ions can also serve to produce static force of attraction within the DNA molecule.
This leads to the folding of DNA into a compact ball-like structure that facilitates its entry into the cell Clark et al. A supercoiled ball like structure of the plasmid will have more chances of entering the competent cell for transformation than the extended open circular form of the plasmid. However, if the size of the DNA approaches the size of the pore, the probability of the transformation decreases sharply.
When using spermidine or other trivalents, the size of the ball-like structure of DNA might exceed the size of pores in the cell membrane, which can be only solved by altering the physical parameters used in the protocol, primarily the heating and cooling cycles. Whether using divalents or trivalents, their concentrations need to be optimized such that all the phosphates of the DNA are not rendered inaccessible, because some parts of the DNA have to adsorb onto the cell surface and for that free phosphates are required, as inferred by Panja et al.
The transformation efficiency is greatly affected by the type of the host cell, as they have different cell surface structures, especially in relation to O-polysaccharides that protrude from the surface of the cell. These surface structures interact with the divalent cations and the DNA, thus making the cell competent for transformation.
Different strains of E. A very dense O-polysaccharide will become a deterrent for the DNA to pass through. However, it has also been claimed that extensive removal of LPS by excessive ethanol-pretreatment reduces transformation efficiency Roychoudhury et al. This can be explained by the aforementioned hypothesis that the DNA first attaches to some external component of the cell membrane, which then assists its movement inside the cell.
Along with the density, the composition of the O-polysaccahride also plays a role in the reception of the incoming DNA molecule Lacks, Therefore, the membrane properties play a major role in DNA adsorption.
The evidences clearly indicate that the physical and chemical treatments used during transformation, i. Magnesium and calcium combinations are seldom used in transformation protocols, the importance of which should be considered.
A combination of divalent and trivalent cations with prolonged incubation times can be suggested to improve the transformation efficiency; as in addition to the charge stabilization, trivalent cations can compact DNA, further aiding its internalization.
Bacterial cells could also be grown in presence of CaCl 2 and MgCl 2 before inducing competency. Heating and cooling cycles used just once in transformation protocols could also be increased to three times for higher transformation efficiencies. These conditions need to be adjusted and optimized for different bacterial species and strains, owing to the differences in their surface properties. Since the natural competency of E. The protocols for preparing competent cells vary by whether transformation is to be achieved via heat shock or electroporation.
In either scenario, a single fresh colony of the desired strain is taken from an agar plate and inoculated into liquid medium for a starter culture Figure 2.
This starter culture and the subsequent larger culture are carefully monitored for active growth by continually measuring optical density at nm OD To obtain high transformation efficiency, it is crucial that cell growth be in the mid-log phase at the time of harvest—which generally occurs at OD between 0.
In all steps, care must be taken to use sterile tools and labware, media, and reagents where appropriate or required. Harvested cells are then processed according to the method of transformation, whether by heat shock or electroporation Figure 2.
The transformation efficiency of competent cells is usually measured by the uptake of subsaturating amounts of a supercoiled intact plasmid e. Cells should not be frozen or stored in liquid nitrogen, as this practice drastically reduces viability. For consistency and to save time, premade competent cells are available in ready-to-use formats from commercial sources.
These competent cells are quality-controlled and tested to meet specifications for transformation efficiency and genotypes. These preparations minimize batch-to-batch variability and significantly simplify the efficient propagation of cloned DNA.
The two most popular methods of bacterial transformation are 1 heat shock of chemically prepared competent cells chemical transformation , and 2 electroporation of electrocompetent cells. The choice depends on the transformation efficiency required , experimental goals, and available resources see competent cell selection. When ready for the transformation step, competent cells should be thawed on ice and handled gently to retain viability. Cells can be mixed by gentle shaking, tapping, or pipetting, but vortexing should be avoided.
Learn the basics of transformation, two types of competent cells, how to perform chemical transformation, and tips for troubleshooting. Note: Negative and positive controls should be included in the transformation step to evaluate the success of the experimental procedure.
With chemical transformation , chemically competent cells are mixed with plasmid DNA and briefly exposed to an elevated temperature, a process known as heat shock Figure 3A. First, cells are incubated with DNA on ice for 5—30 minutes in a polypropylene tube. Polystyrene tubes should be avoided, as DNA can adhere to the surface, reducing transformation efficiency.
Traditionally, 17 x mm round-bottom tubes have been used for best results. Using 1. For smaller volumes of cells in smaller tubes, the heat-shock interval, which depends on the surface-to-volume ratio of the cell suspension, should be reduced. Electroporation involves using an electroporator to expose competent cells and DNA to a brief pulse of a high-voltage electric field Figure 3B. This treatment is believed to induce transient pores in cell membranes, which permit DNA entry into the cells Figure 4.
The most common type of electric pulse in bacterial transformation is exponential decay, where a set voltage is applied and allowed to decay over a few milliseconds, called the time constant Figure 4A.
0コメント