Transformation is a process whereby the genetic materials of a cell are altered by introducing DNA (exogenous DNA) from the surrounding environment through the cell membrane of the organism. It involves the uptake of DNA from either a plasmid or a small fragment of linear DNA by a specific recipient cell. Transformation could occur naturally in some bacteria such as Escherichia coli. There are two types of transformation, natural and artificial transformation. Natural transformation happen when bacteria cells take in DNA naturally through the cell membrane whereas artificial transformation occurs when the recipient cells are forced to take in DNA by chemical or enzymatic treatment (Lorenz & Wackernagel, 1994).
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Transformation occurs in a three step process. The first step is to allow the DNA to precipitate. Cold calcium chloride (CaCl2) is usually added to the mixture of DNA and bacteria because the calcium ion present will neutralise the negatively charged phosphate backbone of DNA (Chan et al, 2013). This is done by ice bathing the samples for 30 minutes to stabilize the bacterial membrane, increasing the between calcium ions and the phosphate backbone of DNA (Li et al, 2010).
Furthermore, heat shock is applied to the cell by incubating the samples in 37°C water bath for 2 minutes. This heat applied could change the fluidity of the cell membrane due to the sudden increase of the temperature (Die et al, 1982). It creates pores in the cell membrane of bacteria allowing the DNA plasmid to enter. Then, cells are placed in ice to prevent the escape of plasmid by closing the pores. The last step of transformation is the recovery phase where L broth is used in order to provide the cells with sufficient nutrients for them to recover.
However, this process takes place only when the bacteria cells are in a state of competence. Competent cells are cells which have the ability to take up foreign DNA from its surrounding environment (Hotchkiss, 2005). Bacterial cells are usually grown to the stationary phase and it will then be harvested for use. This is because bacteria cells at this stage are more competent than other bacteria cells at other stages as it is rapidly dividing producing progeny. Escherichia coli cells are made competent by a process which requires either heat shock or electroporation (Yoo, 2010). In electroporation, an electric filed is applied to the cells to cause in an increase in the cell membrane’s permeability.
The bacteria which will be used in the experiment are the Escherichia coli bacteria. This is because it has the ability to transfer DNA through bacterial transformation allowing the plasmid or genetic materials to spread horizontally through an existing population (Bergmans et al, 1981). Escherichia coli is a gram-negative, rod shaped and facultative anaerobe which is found in the gut. Other than that, most of Escherichia coli strains are non-pathogenic bacteria and can be reproduce very rapidly which is very suitable for lab work. Escherichia coli do not have nuclear envelope surrounding the bacterial chromosome and also contains plasmids which are required in the process of transformation (Sinha & Redfield, 2012).
Plasmid is a circular DNA existing outside the main bacterial chromosomes which acts as a vector. These DNA carries their individual specialized genes for specific functions. In the transformation process, plasmids are used to introduce foreign DNA into the target cells. Some of these plasmids contain the ampR gene, making the particular bacterial cell resistant to ampicillin antibiotic. E.coli cells with the ampR plasmid are known as ampicillin resistant (+ampR) whereas those that does not have this plasmid are known as ampicillin sensitive (-ampR) cells (Adam et al, 1999). The final product of transformation is when the plasmid and the DNA are ligase together and this is called as recombinant DNA.
The aim of this experiment is to transformed Escherichia coli strain into an ampicillin resistance strain using pUC18 DNA. Transformation of competent cells to ampicillin resistance (AmpR) cells involves a series of incubation at different temperature and duration. Apart from that, this experiment is to study and understand the process of transformation occurring in Escherichia coli and also to demonstrate the presence of competent cell. The aim of this experiment is to identify the transformed E.coli cells on a recovery medium and to observe the presence and absence of growth on the L-agar and LAmp agar plates.
MATERIALS AND METHODS:
The materials and methods are shown in the practical manual page number 91 – 94.
Three Eppendorf tubes are labelled 1, 2 and 3 respectively. These tubes are added with components such as transformation buffer (cold), pUC18 DNA, and DNase with the appropriate volume (μL). Tubes 1 and 2 are then incubated in ice whereas tube 3 is incubated at 37°C for 5 minutes. After incubation, the contents of tube 1, 2 and 3 are transferred into tubes labelled 1C, 2C and 3C. These tubes are then placed in the ice for 30 minutes. Then, all the tubes are incubated at 37°C for 2 minutes in the water bath. 200μL of L broth is added to each tube and they are incubated at 37°C for 1 hour in the water bath. A 1:10 dilution in L broth is prepared for 2C. 100μL of the solution in tube 1C is transferred into the L-agar and LAmp agar. This step is repeated for tube 2C-undiluted, 2C-diluted and 3C. All the plates are then incubated at 37°C for 24 hours.
Table 1 : Table 1 shows the presence or absence of growth on both the L-agar and LAmp agar plates for tubes 1C, 2C – undiluted, 2C – diluted and 3C after incubating it at 37°C for 24 hours. The presence of growth is indicated with (+++) for lawn culture, (++) lots of growth and (+) for less growth whereas the absence of growth is indicated with a (-) sign.
GRWOTH ON PLATES:
2C – undiluted
2C – diluted
+++ = Lawn culture shown on the agar plates
++ = Lots of growth presence on the agar plates
+ = Less growth presence on the agar plates
– = No growth
L-agar for all the four tubes, 1C, 2C (undiluted), 2C (diluted) and 3C shows lawn culture (+++) on the agar plates. The L-agar plates show a field or mat of E.coli colonies due to the merging of colonies causes it to be uncountable. However, there is a different in the growth of E.coli for the LAmp agar compared to the L-agar. The agar LAmp for tube 1C and 3C does not show any growth (-) on the plates. LAmp agar plate with the 2C – undiluted sample shows lots of growth (++) presence on the agar plate (>300) whereas the agar plate with 2C – diluted sample shows less growth (+) having 31 colonies presence on the agar.
Recombinant DNA may occur through three basic mechanisms of genetic exchange in bacteria (Etchuuya et al, 2011). Bacterial conjugation is one of the three basic mechanisms for genetic exchange in bacteria. Conjugation occurs by the transfer of genetic materials between bacteria either through direct physical contact or forming a bridge-like connection between cells (Llosa et al, 2002). Conjugation is often referred to as sexual reproduction because it involves the exchange of genetic materials between two bacteria cells. During this process, the F-plasmid or also known as F-factor is being transferred from the donor to the recipient through sex pilus (Griffiths et al, 1999). Once completed, the recipient cell will synthesis a complementary strand. It is a viable donor, therefore having the ability to infect other cells.
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Other than that, the other mechanism involve in the exchange of genetic materials between bacteria is a process called transduction. Transduction involves bacteriophage which is used to transfer the genetic materials from one bacterium to the other (Awwal & Abalaka, 2011). Apart from that, a viral vector can also be used to transfer the gene of interest into another cell. However, using bacteriophage as a vector can be divided into two pathways; lytic and lysogenic pathway. In the lysogenic pathway, the phage chromosomes are incorporated into the bacterial chromosomes and this does not lead to the death of cells whereas the lytic pathway results in the lysis of cell in order to release the phage particles (Court et al, 2006).
Transformation is commonly used by many scientists because it is an easier process to introduce a foreign DNA into bacterial cells and results could be obtained the fastest compared to other methods. Transformation is the process where foreign DNA fragments are transferred into the host cell for growth and replication (Holmes & Jobling, 1996). Firstly, restriction endonucleases such as EcoRI are used to cleave nucleic acid sequence at the GATTC nucleotide to produce a sticky end. Then, DNA ligase is used to ligase the gene of interest to the plasmid. Plasmids and E.coli cells are mixed together and are placed through a series of steps to produce the recombinant DNA.
The first step that occurs in transformation is to allow the precipitation of DNA by adding cold calcium chloride (CaCl2) into the mixture containing both the plasmid DNA and bacteria cell (Chan et al, 2013). In this experiment, the transformation buffer used comprises of CaCl2 where the calcium ion present will neutralised the negatively charged phosphate backbone of DNA by placing the tubes in ice for 30 minutes. Samples are incubated in the ice for 30 minutes permits the bacterial membrane to stabilize resulting in an increase of the interaction between calcium ions and the phosphate backbone DNA (Li et al, 2010). There are other theories stating that the addition of calcium chloride into the samples resulting in the coating of the bacterial surface with calcium ions.
Next, the mixture will be incubated at 37°C for 2 minutes in a water bath. This process is called heat shock as is could change the fluidity of the cell membrane due to the sudden increase of temperature (Froger & Hall, 2007). This creates pores in the bacterial cell membrane to allow the DNA plasmid to enter or even by cell surface invagination. Once the temperature has change the rate of uptake of DNA into the bacteria increase rapidly. Then cells are placed in ice bath immediately to prevent the escape of plasmid by closing off pores. The post heat shock ice incubation step could reduce the thermal motion of plasmid DNA increasing the binding of left over plasmid DNA, to the cell surface which could increase the chances of being taken up by the cell (Singh et al, 2010). The final step in transformation is the recovery phase. L-broth is added into each tube to provide sufficient nutrients for the cells to recover and replicate.
The presence of intracellular restriction endonuclease could decrease the efficiency of transformation. This is due to the fact that different bacteria possess different restriction endonuclease within them (Lopes et al, 2008). However, double stranded DNA could only be used in E.coli because the intracellular exonuclease of E.coli results in a rapid degradation of the DNA (Yu et al, 2000). These intracellular restriction endonucleases will degrade the foreign DNA including the plasmid. Hence, there will be no ampicillin resistance gene the competent cell leading to no transformation.
Table 1 shows, bacterial growths occurred in all the four samples on the Luria Agar (L-agar). The bacterial colonies available on the plates are uncountable because it produces a lawn culture as E.coli could grow naturally in the absence of ampicillin on L-agar. The LAmp agar plates containing samples for tube 1C does not show any growth on it because tube 1C is used as a negative control. It only contains transformation buffer and no DNA has been added to the tube. The absence of pUC18 DNA in this tube results in the absence of any transformed cell because it is not resistant to ampicillin. Therefore, there will be no bacteria colonies present on the LAmp agar plates.
In addition, both the undiluted and diluted samples of tubes 2C show growth of the LAmp agar plates. However, bacteria colonies on the 2C-diluted agar plates are countable whereas the 2C-undiluted is uncountable. This is because tubes 2C displays more competent cells which are presence in the undiluted sample therefore more transformed cells which are resistant to ampicillin are replicated compared to the diluted sample. LAmp agar plates containing samples from tube 3C does not show any growth on it because deoxyribonuclease (DNase) had been added to the sample at the start of the experiment. The function of adding DNase I to tube 3C is to cleave the phosphodiester bonds present in the DNA phosphate backbone hence, degrading the DNA. This results in the formation of small nucleotides.
At the beginning of the experiment, the tubes must be kept on ice to maintain the competence of the host cell in order to keep the membrane rigid. As a result of that, the pores of the cell membrane are kept open. This is to allow the DNase to enter the cell through these pores (Singh et al, 2010). Nevertheless, if the cells are being heated too quickly, the pores will quickly shut preventing the uptake of DNase into the cell. Plasmid DNA is used in this experiment because plasmid occurs naturally in many bacterial cells. Plasmid is able to replicate completely separated from their bacterial genome (Solar et al, 1998). The gene of interest could be easily inserted into a plasmid DNA rather than inserting it into the bacterial genome. Furthermore, plasmid DNA is able to replicate itself in a short period of time given an appropriate condition whereas chromosomes require a long duration of time for it to replicate. Using plasmid DNA will results in a higher of successful transformation.
There are a few limitations that we encountered in this experiment. One of it was the spreading rod which was used to spread the bacteria onto both the L-agar and LAmp agar plates are too hot. This could result in killing some of the bacteria in the sample. Furthermore, contaminations from the laboratory could lead to a different results obtain. This could be minimized by practicing a better technique of the aseptic technique when performing this experiment. In this experiment, it is necessary to wear gloves when handling tubes 2 & 3 because our hands contain nucleases. The use of ungloved hands in this experiment will easily results in DNase contamination affecting the results. Cells which are exposed to DNase would not be able to undergo transformation resulting in inaccurate results. To minimize this error, wearing gloves and changing them frequently during the experiment could also reduce contamination to the samples.
Recombinant DNA technology could benefit many of the industry especially in the medical and agriculture industry. In the medical industry, recombinant DNA may improve cancer research, increase the production of vaccines and other treatments. Recombinant DNA technology allows the scientist to produce human insulin and other growth hormones from bacteria, so are antibiotics that fight against harmful microbes. Apart from that, this technology helps in the development of cancer research by developing many tests to diagnose diseases such as Tuberculosis (TB) (Cederbaum et al, 1984). Other than that, recombinant DNA technology also benefits the agriculture industry by producing genetically modified foods (GMO). These crops are more resistant to insects, viruses and pesticides thus providing a higher yield have better flavours (Pappu et al, 1995). These products have a longer shelf life for transportation and have better flavours and nutritional contents. However, there are many arguments regarding the safety of genetically modified foods (GMO) because it is said that GMO are harmful to our humans. Therefore, some countries do not allow the selling of genetically modified foods.
Bacterial growth occurred in all the four samples placed in L-agar plates whereas only the tube 2C diluted and undiluted shows the presence of bacterial growth on LAmp agar plates. This shows that Escherichia coli have transformed to be an ampicillin resistance strain because of its ability to growth on the LAmp agar. However, tube 1C and 3C does not have any growth on the LAmp agar plates because the cells are sensitive to ampicillin antibiotics. As a result of that, only tubes 2C shows that the cells are competent.
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