Genetic transformation of E. coli by plasmid vector, which carries ampicillin resistanceMolecular Biology Laboratory Report
Name: “Genetic transformation of E. coli by plasmid vector, which carries ampicillin resistance.” (and analys of plasmid vector..?)
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Molecular Biology Laboratory Report
Summary
In our laboratory, genetic transformation of E. coli has been realized by plasmid vector, which carries the ampR gene. As a result, we have been obtained colonies of E. coli which are resistant for ampicillin. The plasmid DNA has been extracted in order to make it more suitable for use in transformation. It has been detected by using Agarose Gel Electrophoresis. The result can be seen below in the text (picture 2). After that, plasmid DNA has been restricted by using plasmid on small fragments. ApaLi, Ban II, Dra I, Pst 1, Eco R 1, BamH 1, Sma I, Sph I, Kpn 1, Hin III restricts enzymes were used for this purpose (see in the table 2).
The 1% agarose gel for electrophoresis procedure has been prepared and the minipreps of DNA and markers ladder have been injected into the start of gel’s tracks. We can see the bands of cuts DNA on the electrophoregramma. We have also calculated the DNA fragments migration distance and the sizes of each fragment with help of the DNA marker ladder (see figure 1.).
Introduction
Bacterial Transformation is the process that occurs when a bacterial cell incorporates foreign DNA from its surroundings, especially plasmid DNA and ends up expressing its gene. Transformation can occur in nature in certain types of bacteria such as Escherichia coli. In molecular biology, transformation is artificially reproduced in the lab via the creation of pores in bacterial envelope and allowing foreign DNA to interact with the host DNA. DNA transfer is made possible by the existence in bacteria’s cell of small stranded DNA called plasmids.
Bacterial plasmids are small double-stranded circular DNA molecule capable of doubling independently of the host chromosomes. Plasmids which are incorporated into the chromosome of bacteria doubles therewith. Many plasmids carry genes that affect the phenotype of the host cell and inform it new properties: drug resistance, the ability to produce toxins for conjugation. In recent years, scientists have described hidden (cryptic) plasmids which have the phenotypic expression. They are found in many bacteria only by ultracentrifugation. Detection plasmids in different species of bacteria showed that the presence of bacterial cells is a widespread phenomenon (1).
Foreign genes cloned into a so-called shuttle vectors. These vectors with equal success are replicated in cells of several masters.
Bacterial cells capable of transfection and transformation are called competent. The culture containing such cells in culture is called step competence. Although the successful completion of the transformation and transfection depends on implementation of many stages, competence in most papers is usually associated with the initial stages which are adsorption and absorption cell DNA (2).
Within the framework of the calcium method, a combination of such effects is used for maintaining the cells in the cold and their treatment with calcium chloride. Chloride calcium can be replaced by barium chloride and rubidium chloride. Incubation at 37 ° C is required for genetic transformation. Calcium ions are only at 37 ° C, i.e. the final stages of treatment of the cells.
Transformation can be performed by plasmid DNA with using the method of freeze-thawing of cells. Transformation efficiency was not lower than in the case of calcium method. The technique reduces to short frozen bacteria cells together with DNA at – 70 o C and subsequent thawing at 37 ° C. DNA absorption is very fast. When defrosting in less than a minute, DNAse treatment did not prevent transformation.
For the construction of recombinant DNA, which contains in its composition the gene to be expressed, adheres to the following policies. DNA is synthesized or isolated cells bearing the genome with the gene fragment which are cloned into an appropriate vector.
Fragments of the genomic DNA are modified by removing from the non-coding regions and neighboring portions of genes. In most cases, the sequencing of the DNA fragment is required (3). Then intermediate constructs recombinant DNA into which the gene is placed under control of the bacterial regulatory elements (promoter, operator, ribosome binding point). These regulatory elements isolated from the hybrid plasmid, constructed especially as a source of regulatory elements. The resulting construct is integrated into a suitable vector.
At present, the bacterium E. coli is the most studied of all existing cells. Bacterial cells that have lost their membrane part (spheroplasts) or all membrane (protoplasts), but retained the cytoplasmic membrane, also can be used for transformation. Transformation of E. coli by plasmid and bacterial DNA characterized some features of adsorption and absorption when compared with DNA transformation of other bacteria and is feasible only in laboratory conditions. Improved methods for the preparation of spheroplasts of E. coli and transfection allowed them to achieve a sufficiently high efficiency of conversion of DNA molecules of different phages (4). Plasmid containing resistance to antibiotic is used to demonstrate bacterial transformation. Competent cells are filtered by growing all the bacterial cells in an antibiotic containing ampicilin. Only the transformed cell will grow in the media while all the other cells which did not incorporate the plasmid containing gene for ampicillin resistance died.
In our work, we use plasmids that carry the ampR gene to transform E. coli cells that lack this gene. A plasmid containing resistance to an antibiotic is used as a vector. The gene of study is incorporated into the vector plasmid. After that, this newly constructed plasmid is then put into E. coli that is sensitive to ampicillin. Then, we identify the transformed cells by adding the ampicillin to the medium. The ampicillin provides a selective platform because only bacteria that have acquired the plasmid can grow on the plate.
The Experimental Procedures
BACTERIAL TRANSFORMATION
Equipment and reagents: Luria broth, 10% bleach, 70% ethanol, water bath at 420celsius sterile, pipette and pipette tips, Petri dishes with antibiotic (ampicilin), marker pen, latex gloves, microbiological spreader, ice and ice buckets (5).
Procedure:
Firstly, sterile 15ml of cell tubes were chilled in ice. It was required to use one tube for each transformation. Frozen competent cells were removed from storage at -800celsius and on ice for 5 minutes. Once the cells had thawed, the chilled pipette tips were used. After that, 1-50ng of ampR plasmid gene were added for ampilcilin resistance in a volume. It was important to ensure that it hadn’t exceeded 10 ul for each 100 ul of competent cell. The tubes were returned in ice for 10 minutes (5). The cells were het shocked for 40-50 seconds in a bath at 420 Celsius. After this stage had been completed, the tubes were immediately placed with cells and DNA on ice. 100 ul of LB media were added. The next stage was to freeze it in ice for 2 minutes. Two plates, one with antibiotic and the other without antibiotic, were inoculated. 50 ul of the mixture from the transformation reaction were applied in each plate. The plates were incubated at 370celsius for 12-14 hours and observation on growth colony was recorded. It was required to wrap it with the filter paper to prevent drying and store in a refrigerator at 40 Celsius. Also a second group of E. coli cells was prepared as a control to verify that E. coli will not grow on agar with ampicillin unless it is transformed and that nothing in the procedure itself affects the survival of E. coli. The procedure was the same for both groups of cells except in the step 4 where ampR plasmids are added to the experimental cells but not to the control cells (6).

PLASMID MINIPREP
Equipments and reagents, luria broth media, resuspension buffer, lysis buffer (SDS detergent, sodium hydroxide (NAOH), precipitation buffer, 10% bleach, 70% ethanol, 100% isopropanol, tris EDTA buffer, microfuge tubes, microfuges, pipettes, latex gloves, vortex, 3.0M potassium acetate, organic solvents (chloroform, phenol) (7).
Procedure
The contents of the culture tube were gently swirled to resuspend the cells. Two 1.5 ml tubes and pipette 1000 ul of the cell suspension were added into each tube. The tubes were placed in a centrifuge and spun for 20 seconds. Withal, tubes couldn’t have been placed on the opposite side of the centrifuge. The supernatant using of a pipette was discarded while the cell residue was not disturbed. The supernatant in a waste container was also discarded. 100 ul of Tris edta buffer (50 mM Tris-HCl, 10 mM EDTA, 100 ug/ml RNAse A, pH 8.0) were added to each tube and the cells were suspended by vortexing. It was very important that the cell suspension was homogenous and no clumps were visible. 200 ul of lysis buffer (1% SDS (sodium dodecyl sulfate), 0.2 M NaOH) were added to each tube. The solutions were mixed and rapidly inverted a few times. Since the chromosomal DNA released from the broken cells could be sheared into small fragments and contaminate the plasmid prep, it was important not to vortex. The tubes were put in ice for 5 minutes. 150 ul of ice-cold Buffer 3 (3.0 M Potassium Acetate, pH 5.5) were added to each tube. The caps were closed and the solutions were mixed by rapidly inverting them a few times. As a result, a white precipitate was formed (8).
The tubes stood for another 5 minutes. After that, they were placed in a centrifuge (balanced) and spin at maximum speed for 5 minutes. The precipitate pelleted along the side of the tube. The supernatants were transferred into clean 1.5 mL tubes without picking up any of the precipitate. The tubes were discarded with the precipitate and the tubes were kept with the supernatant. Before this step had been completed, it was relevant to ensure that a centrifuge was available. An equal volume (about 400 ul) of isopropanol was added to each tube of supernatant to precipitate the nucleic acids. The caps were closed and mixed vigorously. The tubes stayed at room temperature for 2 minutes, and then they were placed with their hinges pointing outward from the center in a centrifuge (balanced) and spun at maximum speed for 5 minutes. This step pelleted the nucleic acids. 200 ul of absolute ethanol were added to each tube and mixed by inversion several times (8). The tubes were spun at maximum speed in a centrifuge for 2-3 minutes with hinges oriented outside. The supernatant was carefully removed and discarded. The tubes were placed in the fume hood with the caps open for 15-20 minutes to dry off the last traces of ethanol. When the ethanol had been gone, 20 ul of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) were added to dissolve the pellet. Pipette was using for pipetting the 20 ul in and out, up the side of the tube to ensure that all of the plasmid DNA came into contact with the TE buffer. The two 20 ul solutions were pulled into one labeled tube and stored in the freezer for future use (7).

RESTRICTRICTION DIGEST
Equipments and reagents, DNA samples, restriction enzymes (Apali, EcoR1,pst 1 etc) stored in 50% glycerol, premade digestion buffer specific for each enzyme to be used, microfuge tubes, microfuge, sterile pipette tips, marker pen, latex gloves, procedure (5).
Procedure:
First of all, sufficient microfuge tubes enough for all restriction enzyme digest were labeled. For the sample to be used, we combined 0.25 µl of restriction enzyme, 1.0 µl 10x digestion buffer and 6.75 µl of deionized water. When pipetting restriction enzyme from the glycerol, it was ensured that only the tip of the pipettes had been dipped into the restriction buffer (9). To the tubes containing the above mixture, 2.0 µl volumes of plasmid DNA were carefully pipetted into each of the restriction digest tubes. The tubes were mixed immediately by pipetting up and down the mixture numerous times. The mixtures were allowed to incubate for 30 minutes at 370celsius. Air bubbles had been successfully avoided at all times to prevent denaturing the enzyme. Surely, a fresh tip was used for every tube. During incubation time, an agarose gel was prepared. The restriction fragments were observed then and analyzed in gel electrophoresis (10).

AGAROSE GEL ELECTROPHORESIS
Equipments and reagents, electrophoresis buffer, ethidium bromide, agarose powder, DNA samples, DNA ladder (1kb) (5).

Procedure:
Agarose was weighed and placed into a conical flask containing 100ml of 1x Tris-AceticAcid-EDTA electrophoresis buffer. The flask was covered with a cling foil at the mouth and a small hole of about 0.5 cm was punctured. The flask was heating in a microwave until the agarose melted completely. The gel was allowed to cool to about 500celsius before casting into a gel tray. To make the DNA visible in the gel, Ethidium bromide was added to the gel solution and the buffer. The gel was placed in the electrophoresis tank containing sufficient amount of1x Tris-AceticAcid-EDTA electrophoresis buffer and the comb was removed. The appropriate volume of the sample had been loaded, about 5 ul in each well after staining with 1 ul of 10000x sybr green. The stained sample was also mixed with 6x DNA loading buffer to a final concentration of x1. Electrophoresis was conducted at120v until the dye front migrated 2/3 of gel. The gel was placed on a ultraviolet transiluminator box and observed with glasses for safety. A photo shot of the bands was taken (11).

Results
After procedure of transferring DNA into E.coli, colonies of E. coli were planted onto the contained ampicillin medium There was colony growth in the petri dish containing gene for antibiotic resistance whereas no growth was observed in the petri dish without ampR. The transgenic cells of E. coli contained gene for resistance to ampicillin were obtained.
The gel for electrophoresis was prepared. The plasmids on the phoregramma and the attendance of plasmid in the miniprep were shown. Further the restrictases were used for cutting plasmid on small fragments. ApaLi, Ban II, Dra I, Pst 1, Eco R 1, BamH 1, Sma I, Sph I, Kpn 1, Hin III restricts enzimes were used for this purpose (see in the table 2).
At the picture 1, we see the DNA bands as result of using Restriction enzyme in order to cut the plasmid. In track 7, DNA ladder (1kb) was used that contains the DNA fragments with the known weights. In track 8, uncut DNA was used. There was colony growth in the petri dish containing gene for antibiotic resistance whereas no growth was observed in the petri dish without ampR.
The Figure 1 illustrates the DNA bands as a result of using Restriction enzyme in order to cut the plasmid.

Discussion
We isolated little amount of competent cells and transferred it into a liquid LB agar media. The next morning the media was thick and cloudy showing that bacterial had grown.
As expected, we have competent bacterial cells with the plasmid vector in which DNA have been transferred. The vector contains an antibiotic resistance gene. Obviously, not all of the cells were successfully transformed. In order to distinguish normal cells from transformants, the presence of the selective agent in the medium is required. In this case, the selective agent is ampicillin. As it was expected (12), cells that have not been transformed were successfully killed. Cells that were duplicated, extracted DNA and checked for the presence plasmid. In order to use a plasmid for further transformations, it was removed. On foregramme cell DNA is shown as a plasmid. So, it proves that everything was done properly.
Then plasmid DNA has been extracted in order to make it more suitable for use in transformation. It has been detected by using Agarose Gel Electrophoresis. The result can be seen below in the text (picture 2). After that, plasmid DNA has been restricted by using plasmid on small fragments. ApaLi, Ban II, Dra I, Pst 1, Eco R 1, BamH 1, Sma I, Sph I, Kpn 1, Hin III restricts enzymes were used for this purpose (see in the table 2).
By digesting the DNA with various restriction enzymes, alone and in combination, the number and relative positions of target sites along the DNA can be determined for each restriction enzyme. This can be used in preparation of restriction maps.
Enzymes of the first type cut DNA at random far from their recognition sequences. They are of considerable biochemical interest, but are rarely used in practice because they do not produce discrete restriction fragments or distinct gel-banding patterns.
Type II enzymes cleave DNA at defined positions close to or near their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns. They are the only class used in the laboratory for routine DNA analysis and gene cloning. Most common are Hhal, Hind iii, Notl, EcoR1.This are the most principal available commercial enzymes.
Some restriction enzymes such as Hae iii and Alul 1 cut straight across the double stranded DNA producing blunt ends whereas enzymes such as EcoR, Bahm1 and Hind iii cut in an offset fashion leaving overhangs of single stranded DNA strands. These are referred to as sticky ends because they are able to form base pairs with any DNA molecule that contains the complementary sticky end.
The number of produced fragments depends on the number of recognition sites in the DNA molecule, each with a precise length and nucleotide sequence.
DNA may be digested with two or more enzymes. However, a buffer that is most compatible with the two enzymes must be used. Many enzyme catalogues have a page that shows enzymes and their compatible buffers. EcoRI and BamHI are effectively used together because of their buffer compatibility. Some other restriction enzymes require special conditions such as bovine serum albumin (BVA) while others require special incubation temperatures.
Restriction enzymes can also express star activities such as cutting at sites which are different from its cognate site. For example, EcoRI is supposed to only cut GAATTC but, under extreme conditions, it might possibly cut CAATTC too. Finally, restriction enzymes’ names are derived from their host organism, the first letter from the genus of the organism and the first two letters from the species name of the organism then followed by the strain or type. Thus, Escherichia coli strain R is EcoR.
After restriction, we must check the obtained fragment. The 1% agarose gel for electrophoresis procedure has been prepared and the minipreps of DNA and markers ladder have been injected into the start of gel’s tracks. We can see the bands of cuts DNA on the electrophoregramma. Also we have calculated the DNA fragments migration distance and the sizes of each fragment with the help of the DNA marker ladder (see figure 1.).

During electrophoresis, the DNA fragments in the sample moved from the sample well through the gel towards the positive electrode that is from the top to the bottom in the picture. Small DNA molecules migrate faster than larger DNA molecules and, therefore, DNA of different band separate into distinct bands during electrophoresis. Ethidium bromide is used to visualize DNA fragments when exposed to uv light.
More DNA in a band gives more intense staining of that band. Large fragments are easily visible while those of low molecular weight are not clearly visible. Bands with same staining intensity and mobility distance are of the same equimolar amounts.
To determine the identity and molecular weight of the fragments represented by the bands, the photograph is compared with a DNA ladder of known identity and molecular weight (1kb ladder).
Each of DNA fragments’ molecular weights were determined by building calibrate standard curves. The results are described in the table.1, table 2, and figure 1.

Conclusion
The cell that grew had transformed whereas the others did not pick up the resistance gene. The plasmid had detected in the prepared miniprep, by agarose gel electrophoresis. The plasmid has been cut by the usage of restrictases. There was colony growth in the petri dish containing gene for antibiotic resistance whereas no growth was observed in the petri dish without ampR.
References
1. Lorenz, M.G., and Wackernagel, W. (1994). Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev., 58 (3), 563-602.
2. D.-M. Zhu, D.-M., and Evans, R.K. (2006). Molecular mechanism and thermodynamics study of plasmid DNA and cationic surfactants interactions. Langmuir, 22 (8), 3735–3743.
3. Bail, G., Filloux, A., and Voulhoux, R (2014). A method to capture large DNA fragments from genomic DNA. Methods Mol. Biol. 10.1007/978-1-4939-0473-0_38.
4. Beninger, R., and Kleber, I. (1971). Transfection of Escherichia coli and Salmonella typhimurium Spheroplasts: Host-Controlled Restriction of Infective Bacteriophage P22 Deoxyribonucleic Acid. Journal of Virology, 8 (2), 192-202.
5. Becker, J., Caldwell, G., and Zachgo, E.A. (1996). Biotechnology: A Laboratory Course, 2nd Ed. New York: Academic press.
6. Activity 4: Transformation of E. coli using green fluorescent protein (2012). APS: Education, 6.
7. Birnboim, H.C. (1983). A rapid alkaline extraction method for the isolation of plasmid DNA. Methods in Enzymology, 100, 243-255.
8. DNA Purification (2012). Protocols & Applications Guide, 8 (12), 1-34.
9. Primrose, S.B., and Twyman, R.M. (2011). Principles of Gene Manipulation, 6th Ed, Blackwell Publishing.
10. Russell, W., Hertz, F., and R. Starr (2008). Biology: The Dynamic Science. CH 18.
11. Barker, K. (2005). At The Bench: A Laboratory Navigator. New York: Cold Spring Harbour Laboratory Press.
12. Sambrook, J., and Russel, D.W. (2011). Molecular Cloning: A Laboratory Manual, 3rd Ed. New York: Cold Spring Harbor Press.

Appendix

Figure 1. Colonies of bacteria that were transformed (on the left) and have not been transformed (on the right) in a medium.

Figure. 2. Agarose gel electrophoresis. A 1kb DNA ladder was used in trak 7.
DNA plasmid. In track, 7 DNA ladder (1kb) was used that contains the DNA fragments with the known weights.
Figure. 3. Restricted plasmid in agagose gel.

standarts
migration (cm) fragment size (bp) log 10 frag size 1/migration (mm-1)
3,5 10000 4 0,285714286
3,6 8000 3,90309 0,277777778
3,8 6000 3,7781513 0,263157895
4 5000 3,69897 0,25
4,2 4000 3,60206 0,238095238
4,5 3000 3,4771213 0,222222222
4,7 2500 3,39794 0,212765957
5 2000 3,30103 0,2
5,6 1500 3,1760913 0,178571429
6,6 1000 3 0,151515152
7,4 750 2,8750613 0,135135135
8,5 500 2,69897 0,117647059
10 250 2,39794 0,1
Table 2. Agarose Gel Electropforetic Anaysis (corresponds to the Figure 2) standart.

Table 3. Agarose Gel Electropforetic Anaysis (corresponds to the Figure 1.) for restriction.
Digests fragment
migration (cm) 1/migration (mm-1) log 10 frag size fragment size (bp) fragment
4,45 0,224719101 3,504827 3197,621429 ApaLi
6 0,166666667 3,0511924 1125,103429
4,85 0,206185567 3,3600019 2290,877674 Ban II
6,35 0,157480315 2,9794083 953,6922615
8,55 0,116959064 2,6627662 460,0088844
9,25 0,108108108 2,5936029 392,2860682
4,35 0,229885057 3,545195 3509,093776 Dra I
7,55 0,132450331 2,7838184 607,8807826
4,45 0,224719101 3,504827 3197,621429 Pst 1
6,1 0,163934426 3,0298421 1071,129833
4,2 0,238095238 3,6093511 4067,720897 Eco R 1
4,2 0,238095238 3,6093511 4067,720897 BamH 1
3,85 0,25974026 3,7784901 6004,683844 Uncut DNA
4,1 0,243902439 3,6547299 4515,750124
4,2 0,238095238 3,6093511 4067,720897 Sma I
4,3 0,23255814 3,566083 3681,99361 Sph I
4,1 0,243902439 3,6547299 4515,750124 Kpn 1
4,25 0,235294118 3,5874626 3867,787185 Hin III

Figure 1.
Figure 1. Graph of migration distance (linear scale) vs. molecular size (logarithmic scale) of the bands in the molecular weight standart.