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The Role of Plasmids in Molecular Cloning

The modern molecular biology have heavy reliance on quick and easy cloning or sub cloning DNA fragments in several plasmid vectors. The plasmids have some relevant features like autonomous origin of replication, conferring to the bacterium in which they reside some form of resistance, and multiple cloning sites. Transformation of cells has a wide uses in the molecular biology development and is a vital tool in genetic engineering. This research project is used in introducing a plasmid that is foreign into bacteria amplifying the plasmid by producing it in large quantities. A plasmid is circular small DNA piece with genetic information that is important for bacterial growth.

 The evolvement of bacteria which has the same environmental growth as that of fungi and mold has made proteins in activating the toxins be produced by other toxins. The sharing of vital information is in the form of genes among themselves. Therefore, plasmids has a role in the transfer of genetic information which is useful to the survival of the bacteria. This research project exploits the features of plasmid for the process of transformation. Taking in of plasmid by the bacteria requires competency taking up the DNA which normally does not pass through the cell membranes of the bacteria. In order to pass through the cell membrane, small holes are created in the cells of the E.coli which is the bacteria used in this project and suspending the bacteria in a high solution concentration of calcium. The DNA are transformed by various procedures discussed in this research.

The key component of this molecular cloning will use the sequences of DNA from the species used in serving the host that is alive for the process of replication of the recombinant DNA and the species to be the source of DNA for cloning. The DNA for cloning was the Green Fluorescent protein genes from the jellyfish. It was treated with enzymes in the generation of smaller fragments of DNA which were later used by combining with vector DNA in generating the recombinant molecules of DNA. The recombinant DNA was then introduced to the E. coli bacteria for generating a population of organisms where the molecules of the recombinant DNA for replication with the host DNA. The vector pGFP which was the donor of the GFP gene contained a marker that was selectable. The gene for the resistance for ampicillin was labeled and coded with B-lactamase enzyme which was used in destroying the ampicillin antibiotic and the visual marker with reporter gene with GFP at 0.07kb from the jellyfish which also was encoded with the green fluorescent protein. Another key component of the research project was the expression vector, Pbcs KS, 3.4 kb which contained a selectable marker called the Chloramphenicol acetyltrasnferrase genes (CAT) which was used in modifying the antibiotic chloramphenicol.

 Week one- understanding the techniques of isolation of recombinant plasmid DNA especially the procedure of transformation by use of heat shock method.

Week two - aimed at understanding the uses of reporter genes in molecular biology by use of experiments that show how screening for the gene of interest.

Introduction of Foreign Plasmids for Amplification

Week three -aimed at understanding how there can be transfer of DNA to a different organism and changing the characteristic of that organism that are observable. It also aimed at becoming familiar with the techniques of sterile and procedure of decontamination that are applicable when handling bacteria by the digestion of plasmids DNA with restriction enzymes. Lastly, the research project aimed at learning how the efficiency of transformation is calculated.

The materials and methods were grouped into two categories and summarized in the table below.

Materials in the lab

Common materials              

The disposal gloves

pGREEN plasmids (0.005 µg/µl)+

Waterproof marker

20 µl micropipette and tips for instructor use only

Safety goggles

Crushed ice

Two micro tubes

Distilled water

6 inoculating loops that are disposable

 Large container with 10% bleach solution containing all the used disposable pipets and loops and for sterilizing the petri dishes

Micro tube containing 50 mM CaCl2

Plates with E.Coli cells streaked out and grown overnight

2 plates labeled LB medium #

Squit bottles containing 10% bleach

Foam cup containing crushed ice

UV light

Bacterial waste container

Water bath containing floating tube racks

6 pipets that are disposable

37 degree Celsius incubator

2 plates with LB medium ampicillin

parafilm

Micro tube rack

Micro tube filled with Hindlll

2 plates containing LB medium+

The enzymes- This research project used the restriction enzyme Hindlll and EcoRI.

DNA- the DNA used in this project research was T4 DNA ligase.

Techniques- several techniques like the miniprep, electrophoresis and ethanol precipitation were used. They are discussed below for each of the project experiments carried out.

The DNA sequence analysis was used in the cloning strategies. The use of plasmids is common as the plasmids used are multicopy plasmids that can be purified from small volume cultures. The alkali lysis technique is commonly used technique in enabling the plasmid DNA to purify enabling further manipulation.  The first experiment named project 1 was for the transformation of the bacteria with the vectors designed to prepare for electroporation. For the media with the growth for bacterial culture, the antibiotic ampicillin was used. There is centrifugation for pelleting the cells of E.coli and the media supernatant is then thrown away. The cells are re-suspended in a solution containing buffered glucose. A strong alkaline solution of NAOH containing the sodium dodecyl sulphate which is used as a detergent is added in lysing the individual bacteria and thereby releasing the plasmids that are buy then supercoiled.

The first lysis is limited by not being allowed to go long enough in unwinding the DNA plasmid even though the cell proteins have to get enough time to denature and disrupt the membrane of the cells.  The plasmid DNA that is in the supermant is then purified further or is precipitated by adding ethanol. Since there is already a high concentration of salt in the solution as a result of the added potassium acetate for neutralizing the sodium hydroxide, it is advised to take note of the high concentration. The antibiotic ampicillin 100 µg/ml was used for the growth of the pGFP and for the pBCKS the antibiotic used was chloramphenicol in the media of 30 µg/ml. The GFP gene was isolated from the vector Pgfp which had already been introduced into MCS region containing the pBCK KS that was controlled by pluck promoter. The cells of the recombinant E.coli that was generated containing Pbc ks –GFP vector were then identified by their growth on the antibiotic chloramphenicol and also the bright fluorescent under the UV light. The plates of LB and LB/AMP are added to an antibiotic ampicillin and stored in refrigeration until they were ready for use. The illustra plasmid prep mini spin kit was designed for rapid extraction of the plasmid DNA and the purification of plasmid DNA from the 1.5 ml and 3 ml cultures of E.coli. The procedure was completed in 9 minutes where two cultures of plasmid DNA were yielded with quality and purity compatibility. The techniques included cloning, restriction enzyme digestion, DNA sequencing and PCR amplification.

The Importance of Transformation in Genetic Engineering

The next procedure was the lysis where the cell suspension was added to the 175 lysis buffer type 7 for the bacterial pallet for re-suspension. The cell re-suspension was achieved by pipetting up and down to increase the DNA recovery. The lysis buffer type 8 was added to the mix by gentle inversion for 4 minutes as the lysis buffer type 8 contains NaOH that when used on prolonged incubation results in denature of the plasmid DNA. Neutralization was done by adding lysis buffer type 9 to the mix by gentle inversion until there was even dispersion of the precipitate. The next process involved the centrifugation where each purification was performed by placing one illustra plasmid mini column in one collection tube.

The antibiotic ampicillin 100 µg/ml was used for the growth of the pGFP and for the pBCKS the antibiotic used was chloramphenicol in the media of 30 µg/ml. The GFP gene was isolated from the vector Pgfp which had already been introduced into MCS region containing the pBCK KS that was controlled by pluck promoter. The cells of the recombinant E.coli that was generated containing Pbc ks –GFP vector were then identified by their growth on the antibiotic chloramphenicol and also the bright fluorescent under the UV light. The plates of LB and LB/AMP are added to an antibiotic ampicillin and stored in refrigeration until they were ready for use. The illustra plasmid prep mini spin kit was designed for rapid extraction of the plasmid DNA and the purification of plasmid DNA from the 1.5 ml and 3 ml cultures of E.coli. The procedure was completed in 9 minutes where two cultures of plasmid DNA were yielded with quality and purity compatibility. The techniques included cloning, restriction enzyme digestion, DNA sequencing and PCR amplification.

The next procedure was the lysis where the cell suspension was added to the 175 lysis buffer type 7 for the bacterial pallet for re-suspension. The cell re-suspension was achieved by pipetting up and down to increase the DNA recovery. The lysis buffer type 8 was added to the mix by gentle inversion for 4 minutes as the lysis buffer type 8 contains NaOH that when used on prolonged incubation results in denature of the plasmid DNA. Neutralization was done by adding lysis buffer type 9 to the mix by gentle inversion until there was even dispersion of the precipitate. The next process involved the centrifugation where each purification was performed by placing one illustra plasmid mini column in one collection tube.

Q1: The color of the Pgreen DNA plasmid when exposed to ultra-violet light fluoresce

The table below summarizes the results obtained.

Plate

Actual change

Expected  change (control)

LB –plasmid

White colonies were observed

Visible cells should fluoresce

LB +plasmid

No growth

No growth (No DNA)

LB/Amp – plasmid

White colonies observed

White colonies

LB/ Amp + plasmid

Several individual colonies

Several colonies that fluoresce on exposure to ultra-violet light

Q2: Not all the plates had the bacteria growing on them

Q3: On observing the results on each plates; the images were recorded below;

  1. LB –Plasmid
  2. LB +Plasmid
  3. LB/Amp – plasmid
  4. LB/Amp + plasmid

Plates

Actual results

Expected results

size

LB –Plasmid

Colonies formed with no fluoresce

Undigested pGFP

3800 bp

LB + Plasmid

No colonies

Digestion of pGFP

2000 bp

LB/Amp - plasmid

Growth

No digestion of pBCKS

300 bp

LB/Amp + plasmid

Colonies formed and the cells fluorescence

Digestion of pBCKS

4000 bp

A comparison of the gel electrophoresis of the result and that of the control indicated that the plasmid DNA existed in linear and circular forms. While the control should exist in three different forms, supercoiled, open circular and linear DNA. The plasmid digestion results were observed to be complete in plate LB –DNA, partial in LB/Amp + plasmid while no digestion in LB/Amp – plasmid.

Using GFP Gene for Recombinant DNA Production

Plate with no DNA and ampicillin plated control cells had visible cells which did not fluoresce. Therefore, white colonies were observed. A smear like layer was observed in the cells. This was because the cells of the host bacteria are viable when ampicillin is not present. The pGREEN plasmids contain a GFP version that is mutant and is linked to a different gene referred to as beta-galactosidase. The gene mixture contain protein that is produced when combined to form yellow-green bacteria that can be observed under normal light. However, when colonies are exposed to UV light, they fluoresce. The plate with no DNA and ampicillin had partial digestion. The results obtained only had partial digestion instead of the expected full digestion.

The plate labeled LB +Plasmid had no growth. The lack of growth was because the cell of the bacteria are sensitive to ampicillin. In the absence of Pgreen plasmids, the cells cannot resist to ampicillin. The plate with DNA plasmid with no ampicillin coated cells formed white colonies which formed a smear layer of cells. This indicated their full digestion. Since both the cells that were transformed and those not transformed are viable when ampicillin is present, the growth observed was majorly from the cells not transformed which overshadowed the cells transformed resulting to fluorescent cells. The E.coli that is used in the research study in the process of transformation lives in the gut of human beings. The plasmids allow sharing of genes among themselves and this makes them naturally adjust to the new environment. The cell transformation is based on the ability of the bacteria in maintaining the plasmids and replicating them during cell mutilation. The plasmids are useful in events transformation in bringing interested DNA into the cell where integration into the genome can occur or it can have no change within the bacteria. When it remains into the bacteria, it can then be translated into the rare proteins that is not existing in that organism.

The plate labeled LB/Amp + plasmid had different colonies which fluoresce on exposure to the ultra-violet light. The observed colonies was because there was transformation of cells which were resistant to the ampicillin and resulted to uptake of the DNA Pgreen. The cells of the bacteria that were not transformed did not grow when the ampicillin is not present. The fluorescence observed originated from the green fluorescent protein that had been encoded by the plasmid. The usual location of the green fluorescent protein in jellyfish is at the bell margin. Therefore, when certain conditions exists, they fluoresce. Inserting the green fluorescent into a plasmid after using the transformation procedure, enables the bacteria transformed to express the gene acquisition and production of fluorescent protein that causes the green glow on exposure to UV light.

The plasmid contains the resistance gene –ampicillin. Ampicillin works as an antibiotic by preventing the bacteria from forming cell walls which can kill the bacteria. The presence of ampicillin resistance gene helps in directing enzyme produced by blocking the ampicillin from acting. In this way the survival of the bacteria is enhanced. Bacteria present will be without plasmid and with the resistance gene will not be able to have growth on the plate as there is ampicillin in the medium. Therefore, only the transformants will be able to survive. The green fluorescent protein is used in this research as molecule reporter. The aim of understanding the recombinant technique and procedure of transformation by use of heat shock method was achieved. From the research project, understanding of the screening of the gene was achieved as the importance of marker genes were observed in the experiments. In addition, the investigation of the transfer of DNA to another different organism was achieved as the screening enhanced observation of the physical characteristic that resulted from the transfer.

Materials and Methods

The control plate in the research project were plate number one and plate number two for positive control and plate number 3 for negative control. The three different plates were giving tests for different component combination of the experiments in verifying the expected functioning. The plates with the transformants was the plate for experiment and was the plate number 4. This was because the plate contained the antibiotic whose function was to allow only the growth of bacteria with pGREEN plasmid and growth of the gene for antibiotic resistance.

When the UV light was shined on the plate containing the transformants, the color of the bacteria was expected to have no visible fluorescence. On comparison with the observation made when the light was shined not the pGREEN plasmid, there was some difference. The colonies of the bacteria that was transformed glow green when light was shined on them. However, when the UV light was shined on plasmid in the tube, there was no fluorescence visible. The reason for the difference was because the plasmid has no reaction with the UV light shines but the reaction is caused by the protein that is formed upon translation of the plasmid. The production of the protein for reaction only happens when there is incorporation of the plasmid into the bacteria.

On the plate with no DNA and ampicillin, there was no presence of antibiotic, therefore, the bacteria being wild type grow normally thereby forming a lawn across the whole plate. On plate two the bacteria was not transformed and therefore not able to grow with the antibiotic present. This was because the cell walls of the bacteria were not formed leading to the death of the cells. On the fourth plate, any bacteria that has undergone transformation was in taking the plasmid of Pgreen. Therefore, they were enabled growth as the antibiotic was present. Moreover, the plasmid also contained the gene for resistance by antibiotic and the bacteria therefore incorporated the plasmid into their cells. The bacteria therefore was able to fluoresce green.

References

Brown T (2006). Gene cloning and DNA analysis: an introduction, Blackwell publishers, ISBN 978-1-4051-1121-8.

Cohen SN, Chang AC, Boyer HW. (1973). Construction of biologically functional bacteria plasmids in vitro, proceedings of the national academy of sciences of the United States of America, vol. 70(11), pp. 3240-3244.

Garret, Grisham (2010). Biochemistry, Cengage learning, p. 380.

Higuchi R, Bowman B, Freiberger M, Ryder (1984). DNA sequence from the guagga, an extinct member of the horse family, journal of nature, vol. 312(5991), pp. 282-284.

Jackson DA, Symons RH, Berg P (1972). Biochemical methods for inserting new genetic information into DNA of Simian Virus 40: circular SV40 molecules containing lambda phage genes and the galactose operon of Escherichia coli, proceedings of the National Academy of science, vol. 69(10): 2904-2909.

Lederberge J (1994). The transformation of genetics by DNA: an anniversary celebrating of Avery, MacLead and McCarty, journal of genetics, vol. 216(1); pp. 423-426.

M Grisham, Charles (2013).Biochemistry. Cengage learning publishers, ISBN 978-1133106296.

Oldernbur J, Dolan G, Lemm G (2009). Hemophilia care then, now and in the future, journal of Haemophilia, pp. 2-7.

Patten CL, Glick BR, Pasternak J (2009). Molecular biotechnology: principles and applications of recombinant DNA, Washington DC, ASM press, ISBN 978-1-55581-498-4.

Pfeifer A, Verma IM (2001). Gene therapy: promises and problems, annual review of genomics and human genetics, vol.2; pp. 177-211.

Watson JD (2007). Recombinant DNA: genes and genomes: a short course, San Francisco, ISBN 978-0-7167-2866-5.

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