Genes Transformation

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Sophomore (College 2nd year) ・Biology ・MLA ・4 Sources

Genes can be transformed. Genetic transformation is the alteration of a mobile resulting from direct absorption and incorporation of exogenous genetic materials in the neighboring surroundings through the cell membrane. For genetic transformations to take place, a recipient micro organism need to be in competence i.e. be capable of taking up overseas DNA in environmental conditions such as cell density, hunger and can also be induced in a laboratory by using providing the necessary surroundings (Bailey et al. 2013).
Genetic transformation can occur in a number of methods on condition that the necessary surroundings is provided, ways in which genetic transformation can be arrived at include; electroporation, projectile bombardment, and heat shock (Bailey et al. 2013).

Electroporation

Electroporation involves the process where cells are bathed in a liquid medium that contains foreign DNA and then subjected to pulses of electricity which increases the permeability of the plasma membrane hence causing the cell to take up foreign DNA from the neighboring liquid medium (Kotnik et al. 2015).

Heat Shock

The heat shock process of genetic transformation involves cells being exposed to a sudden increase in temperature which in turn increases the permeability of the cell plasma membrane and makes it absorb foreign DNA from the neighboring medium (Bailey et al. 2013).

Projectile Bombardment

The projectile bombardment process is used to achieve genetic transformation by bombarding cells using tungsten pellets that are coated with foreign DNA by using a gene gun. The bombardment caused by the gene gun caused the cell to take up DNA from the tungsten pellets.
Genetic transformations have widely been employed in areas of biotechnology i.e. in the field of medicine where diseases caused by defective genes are treated by gene therapy a process where defective genes are replaced with healthy copies (Bailey et al. 2013).
In the field of agriculture genetic transformation is employed to help crops improve disease resistance, and frost or drought tolerance, this is achieved by introducing necessary lacking traits into a particular plant species. Bioremediation also employs genetic transformations to help in digesting oil spills by introducing certain bacterias (Bailey et al. 2013).
My exercise will employ the heat shock process of genetic transformation to deliver a vector containing DNA to aid gene transfer. Common vectors used in gene transfer include the phages and plasmids.

Phages

Phages are viruses that infect bacteria, genes of interest are added to a phage genetic component and then used to infect the bacteria. During infection, phage particles induce their genetic material incorporating the gene of interest into the host cell bacteria causing the transformation (Estienne et al. 2015).

Plasmids

Plasmids are circular double stranded DNA’s that contain genes beneficial to the bacterial cell containing it. Bacteria containing plasmids revolve them around to make sure that the plasmid’s beneficial genes are shared all around the bacteria as they assist in bacterial adaptation to new environments (Estienne et al. 2015).
My exercise will major on the transformation of E.Coli bacteria with a gene code for green fluorescent protein(GFP) under certain conditions and see whether the transformation will be successful. The gene originates from bioluminescent jelly fish (Aequorea victoria). The GFP causes characters to fluoresce and glow in the dark but only in the presence of sugar arabinose which acts as a switch in glowing or not.
The presence of sugar is necessary for glowing as it binds the GFP biosynthesis pathway. In my experiment, I will use a specially designed plasmid pGLO as a transformation vector. The pGlO plasmid contains the GFP gene as well as an ampicillin resistance gene which is an antibiotic (Bailey et al. 2013).
The ampicillin is necessary for screening for the cells that took up the plasmid (transformed) by plating the cells on a media containing ampicillin. Only cells that took up the plasmid will survive and eventually grow on the media containing ampicillin.
In the construction of pGLO plasmid and GFP expression, the DNA code for the pGLO plasmid is engineered to include a part of the arabinose operon, and both the araC gene and the promoter are made present. The remainder of the arabinose operon is removed and replaced with the GFP gene hence in the presence of arabinose the araC gene promotes RNA polymerase binding, and GFP is produced. The significance of this study is that it boosts knowledge on genetics, a field which is majorly applicable in our daily lives today as explained above where is is used in the field Agriculture and Medicine.

Methods

Micropipette Practice

A micropipette is a device used in a lab experiment to add little samples at a time. The volume adjustment wheel is rotated to set the volume. The minimum accurately volume to be pipetted using this micropipette is 100ml, and the maximum is 1000ml (Rehm et al.2013).
Use the volume adjustment wheel to set the pipette at 250 µl mark and open the microcentrifuge tube with food coloring then empty the microcentrifuge tube and place the tubes in a tube rack.
Open the box holding the pipette tips and be very cautious not to touch the tips as this may cause contamination leading to wrong results. Firmly press the pipetter into the tip and remove it from the box (Rehm et al. 2013).
Hold your pipette tip above your food coloring sample and depress the push button down to the first stop and hold it firmly in the position as your gradually lower the pipette into the solution to a depth of 2 to 4mm for a duration of 2 to 3 seconds.
Release the push button slowly to its initial position then give he solution a few seconds to be drawn into the pipette tip and then remove the tip from the solution while keeping the pipette in a vertical position (Rehm et al. 2013).
In transferring the solution to the empty microcentrifuge tube, place the tip to the wall of the empty tube and press the push button to the first stop and then press it again this time to the second stop. While still holding the push button down, wipe the tip against the wall of the tube to remove any hanging drops on the tip.
Remove the tip from the tube then release the push button then hold the pipette over the used tip container and push the tip ejector button. Pipette different volumes of food coloring, for example, try using 500µl and 1000µl volumes of food coloring.

Procedure

In performing this experiment procedure, the steps described below should accurately be followed as described. Precision is important in attaining the right expected results as a lack of accuracy will lead to results different from those expected (Rehm et al. 2013).
First, obtain two microcentrifuge tubes and label them using initials, one tube should be labeled +pGLO and the other tube labeled –pGLO. Find a pair of vinyl gloves that fit you that should be worn all through the experiment due to the handling of bacteria along the experiment process.
While using a micropipette with a fresh tip, transfer 250µl of transformation solution into each of the two tubes. The transformation solution should contain calcium chloride to enhance the permeability of the plasma membranes hence increasing bacterial cells competency and the efficiency of successful transformation (Bailey et al. 2013).
Close the tube with lids and put both the tips on the beaker containing ice then using a sterile loop, pick a bacteria of a single colony from the starter plate, pick the +pGLO tube and insert the loop into the transformation liquid at the bottom of the tube.
Spin the loop ensuring the solution does not spill out until you ensure that the entire bacterial colony is equally dispersed in the transformation liquid ensuring that no lumps are present then return the tube back to the ice bath to cool. Used loops should be disposed to avoid confusion leading to contamination.
Using a new sterile loop to avoid contamination, pick the -pGLO tube and insert the loop into the transformation liquid at the bottom of the tube. As previously done for the +pGLO, spin the loop until the bacterial colony is equally dispersed and put the tube in an ice bath to cool (Rehm et al. 2013).
Obtain a new sterile loop and immerse it in the pGLO plasmid DNA marked tube then remove a loopful of plasmid, you will find a thin film of solution across the ring. Insert the loop of the plasmid into the +pGLO tube and mix the plasmid into the transformation fluid and the E.coli suspension. Be cautious not to add pGLO plasmid to the –pGLO as there should be no pGLO in the tube as it’s labeling suggests.
Incubate the tubes on ice for around ten minutes; this is a crucial step in the experiment as successful transformation of pGLO plasmids takes place by sticking to the plasma membrane of the competent cells (Rehm et al. 2013).
While the tubes are on the ice, obtain Luria Bertani broth (LB) and agar plates from your teaching assistant. Ask for 1LB plate, 2LB/amp plates and 1LB/amp/ara plate and label the bottoms of the plates as shown below. Note that ampicillin present in aga media; ara is equal to arabinose present in aga media.

Label the Lb plate -pGLO
Label one LB/amp plate -pGLO
Label the other LB/amp plate +pGLO
Label the LB/amp/ara plate +pGLO

genes_transformation_1

After 10 minutes on the ice, place both the tubes in a floating rack of water at 42°C for exactly 50 minutes then transfer them to hot water to perform the heat shock experiment which enhances the uptake of the plasmid into the E.coli cells.
The uptake of the plasmid by the bacterial membrane causes the bacterial plasma membrane to be more permeable to the plasmid solution hence increasing their competency and the rate of successful transformation (Bailey et al. 2013).
After 50 minutes of the heat shock treatment, immediately place both tubes back into the ice (make sure the ice beaker is ready and close when removing the tubes from the hot water to improve accuracy) and incubate the tubes on ice for 2 minutes.
Remove the tubes from the ice after 2 minutes and place them on a rack then using a fresh tip add 250 µl LB nutrient broth to the +pGLO tube. Using another fresh tip add 250 µl LB nutrient broth to the –pGLO tube and then close the tubes and incubate them at room temperature for 10 minutes.
Flick the tubes with your finger after 10 minutes to mix the contents then using a fresh tip for each tube, pipette 100 µl of the transformation (+pGLO) and control (-pGLO) suspensions into the suitable LB nutrient agar plates.
Using a new sterile loop for each plate, spread the suspensions until they are evenly distributed around the agar surface by quickly skating the agar surface of the loop back and forth across the plate. Do not press into the agar deeply (Rehm et al. 2013).
Stack up all your plates and tape them together and turn the stack upside down then label them with identity, give your teaching assistant the stack to place it in a 37°C incubator for 24 hours. The time should be strictly observed.

Results

The results should be observed after a week under normal lightning then the lights should be turned off to observe the plates under UV light.

Observations on Transformation Plates

On the transformation plates (+pGLO/LB/amp) plate, a bacterial growth of around 25% was witnessed, 13 bacterial colonies were found by counting the number of dots that were present on the plate. The bacterial colonies were green in color under normal light conditions and UV light.
On plate (+pGLO/LB/amp/ara), a bacterial growth of 50% was witnessed, and there were 74 independent bacterial colonies. The bacteria color was green under normal light conditions and glowed green under UV light.

Observations on Control Plates

On plate (-pGLO/LB/amp), no growth was noticed, and the bacterial colonies under normal and under UV light were clear in color. On plate (-pGLO/LB), there was a 90% increase with no individual colony on the plate. The plate had no bacteria but had a clear film both under normal lighting and under UV light.

Discussion

The plate –pGLO shows that non-transformed E.coli will grow on a plain LB plate with no ampicillin as it needs ampicillin to grow. The plate –pGLO/LB/amp shows that nontransformed E.coli will not grow on LB plates with ampicillin (Bailey et al. 2013).
The plates +pGLO/LB/amp and –pGLO/LB/amp are used in comparison to determining if any genetic transformation has occurred for the reason that one plate has the gene and one does not. The heat shock made the E.coil plasma membrane more permeable hence increasing the competency of cells (Bailey et al. 2013).
The glowing bacteria tells that the attempt to genetically transform E.coli cells with pGLO plasmid was successful. The three factors that must be present in the bacteria environment in order to see the glowing green color include the arabiose sugar, araC gene, and GFP (Bailey et al. 2013).
The ability to turn on and off particular genes in an organism gives the organism the advantages of adapting to different environments which increase the organism's chances of survival in new or changed environments (Bailey et al. 2013).

Works Cited

Kotnik, Tadej, et al. "Electroporation-based applications in biotechnology." Trends in biotechnology 33.8 (2015): 480-488.
Rehm, Heidi L., et al. "ACMG clinical laboratory standards for next-generation sequencing." Genetics in medicine: official journal of the American College of Medical Genetics 15.9 (2013): 733.
Swart, Estienne C., and Mariusz Nowacki. "The eukaryotic way to defend and edit genomes by sRNA‐targeted DNA deletion." Annals of the New York Academy of Sciences 1341.1 (2015): 106-114.
Tille, Patricia. Bailey & Scott's Diagnostic Microbiology-E-Book. Elsevier Health Sciences, 2013.

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