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Describe the cloning strategy that was used in this experiment. What is directional cloning and what are its benefits?
Cloning: The process of producing genetically identical fragments or indivuals from a desired DNA or organism is called cloning.
It is of three types.
The cloning strategy used in this experiment was gene cloning.
Gene cloning: This is a multistep process in which identification, isolation and purification of a desired and analysable gene fragments of an organism are used in cloning.
This experiment involves two types of DNA.
Donor DNA: This is a source for gene of interest.
Vector DNA: The desired DNA fragment is transformed through this vector DNA. This DNA is usually circular (plasmid).
The DNA which consists of segments of different origin in its structure is called recombinant DNA. Exemplifying, the bacterial cell which has both vector DNA and mammalian donor DNA in its genetic material is called a recombinant bacteria.
The donor DNA and the vector DNA are treated with the restriction endonucleases producing compatible staggered ends which are combined with DNA ligase enzyme later and are introduced into bacterial cell via transformation.
In this experiment the DNA fragment that is to be identified and isolated is PTEN (human tumour suppressor) Gene. This acts as donor DNA which is identified and isolated and later amplified by polymerase chain reaction (PCR cycle).
pET blue is taken as a vector gene.
These two DNA molecules are digested with the restriction endonucleases such as BstE II, EcoRI, EcoRV and HincII.
The cohesive ends of the vector and target DNA are bound using the DNA ligase by forming phospho - diester bonds. The recombinant gene product is transferred to the competent cells of bacteria cultured in Sterile L-broth (here 1% tryptone, 0.5% yeast extract, pH 7.4, 0.5% NaCl).
This type of process in which non complementary sticky ends are produced and are ligated in a specific direction preventing the vector from recircularisation or self ligation is called DIRECTIONAL CLONING.
As the cloning is directed it is named as directional cloning. The advantages of directional cloning are:
It is convenient to produce recombinant DNA molecules without any confusion in fragments.
Minimise self-ligation by producing heterologous ends with the help of two restriction enzymes.
2) Draw a diagram of the PCR cycle indicating how restriction Sites can be incorporated at the ends of amplified DNA?
Polymerase chain reaction allows the specific and exponential synthesis of predetermined DNA region via the use of two small , specifically determined fragments of DNA(Primers or Oligo nucleotides). [Elizabeth van pelt Verkuil and Alex van Belkum, 2008].
As the two primers are used (one for each strand of DNA), primer hybridisation (annealing) and dissociation directs the DNA amplification to be in 5' - 3' direction in both strands. The primers used here acts as Okazaki fragments. For PCR technique the following are essential:
Two Oligonucleotide primers.
Taq polymerase (terminal transferase activity).
MgCl2 (Mg2+ influences the productivity and fidelity of polymerases), forward and reverse primers.
5' CCA GTG TTG ACA GCC ATC ATC AAA GAG ATC 3'
(HincII site in bold)
5' GGC TCG AAT TCA GAC TTT TGT AAT TTG TGT ATG 3'
(EcoRI site in bold)
A typical PCR reaction cycle follows the following three temperature dependent steps:
DENATURATION: double stranded DNA molecules are denatured into single stranded DNA which acts as templates for further amplification at 95Ëšc.
ANNEALING: Hybridisation of oligonucleotide primer to each strand is done by lowering the temperature to 45Ëšc-65Ëšc which is the optimal temperature (Tm) for annealing (attachment).
ELONGATION: elongation of DNA occurs from beginning of 3'end and follows in 5'-3'direction by maintaining the temperature at 72Ëšc.
Number of cycles is proportionate to amount of target DNA strands are produced exponentially at the rate of 2n, where n stands for number of PCR cycles (Rod Reed 2003, second edition).
The cycling parameters in this experiment are as follows:
The initial denaturation of template DNA is for 10 minutes at 95Ëšc.
The Denaturation , Annealing and Extension is cycled at 30 times at 95Ëšc , 55Ëšc and 72Ëšc for 15 sec, 60 sec, 60 sec respectively.
The unfinished extensions can be completed by cycling 30 times at 72Ëšc for 10 minutes.
Fig: Genetic engineering concepts
3. Discuss the promoters present in the PETblue-1 plasmid
pETblue-1 vector is used to identify the desired fragments by using Blue-White Screening method. The promoters present in the pETBlue-1 plasmid are:
T7lac Expression promoter
E.Coli (tet) promoter.
Blue/white screening is independent as the T7lac promoter is in opposite orientation to E.coli promoter which mediates the screening method. In this experiment the lac operon is stimulate dwith IPTG and an intact vector and the two coded halves of Î²-galactosidase enzyme combine to form a functional Holoenzyme which hydrolyses X-gal (Lactose used in this experiment) to generate blue colour. On the other side when these target gene sequences are inserted into multiple cloning site(MCS),it disturbs the expression of lacZ Î±-peptide in a lacZ sequence of E.coli when plated in presence of X-gal which makes the IPTG-treated colonies to stay in white color. This is because T7lac promoter expression to be cloned in opposite to the tet promoter, if not the basal expression of target sequences are absent. Thus T7lac protein orientation in opposite direction controls protein production and also controls blue/white colonies. pETblue-1 facilitate unfused, native protein expression, that must encode at 5"end. ECORV cloning site(GATATC) is optimally positioned to T7lac promoter gene to bind to ribosomal binding site(RBS) by which ATG start codon or g-nucleotide at 5"end will create an optimal E.coli translation initiation site. Hence pETblue-1 down primers are used for performing single stranded sequencing.
The two origins of replication analysed in this experiment are
(i) f1 origin which produces single stranded vector under desired conditions.
(ii) Conventional origin of replication.
4) Discuss the approaches that can be applied to confirm the presence of the PTEN insert in the pETBlue-1 after transformation
The presence of PTEN insert in the pETBlue-1 after transformation can be explained by BLUE/WHITE SCREENING.
This system takes advantage of the fact that a functional Î²-galactosidase enzyme can be generated from separate N-terminal and C-terminal fragments of the enzyme protein. This is called insertional inactivation of an enzyme coding gene. The antibiotic resistance vector like AMPr and TETr contain lacZ gene which shows the presence of Î²-galactosidase which converts lactose, a disaccharide to glucose and galactose. This lacZ gene is usually present on E.coli chromosome, but some of them lack lacZ gene portion hence it can be a complete, functional Î²-galactosidase when lacZ portion filled with a plasmid.
In this screening method, X-gal is used as lactose to identify the presence of functional Î²-galactosidase and an inducer IPTG were added to the growth medium. A blue colour was observed when X-gal is broken by Î²-galactosidase which shows that the competent cells contain plasmid with an uninterrupted lacZ gene. White colour was observed when lacZ gene interrupted by foreign DNA hence X-gal was not broken by Î²-galactosidase( foreign DNA inactivated lacZ gene by insertion)
COLONY HYBRIDISATION PROBE:
This hybridisation technique was used for the identification of recombinant PTEN gene. The colonies are transferred to a nitrocellulose or nylon membrane which is treated to remove all contaminated materials except DNA which even can denaturate DNA. To this membrane specific labelled denatured probes were added to promote nucleic acid hybridisation. Later the membrane is washed to remove the unbound probes from the membrane and the bound probes indicate the presence of inserts (Brown, 1998 p382).
Agar, x-gal, IPTG
Blue Colony (non-recombinant)
White Colony (recombinant)
Blue colonies- Î²-galactosidase synthesised, X-gal is present.
White colonies-Î²-galactosidase not synthesised, X-gal is absent.
5. TRANSFORMATION RESULTS:
The transformation shows the result that PTEN is inserted in pET blue vector is transformed in E.coli cells
Bacterial growth on each plate:
Transformation and ligation was performed in the experiment.
N (NEGATIVE CONTROL): This plate contains only agar medium hence no colony was observed at both concentrations due to the absence of plasmid.
RC (RECOMBINANT CONTROL): This plate contains 45 white colonies at a concentration of 5ÂµL and 110 white colonies at a concentration of 50ÂµL i.e.; the colonies present are recombinant.
NRC (NON-RECOMBINANT CONTROL): This plate contains 30 blue colonies at a concentration of 5ÂµL and 150 blue colonies at a concentration of 50ÂµL as they turned into blue colour it proves that transformation has occurred i.e.; X-gal is present and broken by Î²-galactosidase.
L (LIGATION): This plate contains 10 white colonies at a concentration of 5ÂµL and 34 at 50ÂµL respectively shows that ligation is done successively. The recombinant plasmids when transferred into host cells the lacZ gets interrupted with foreign DNA and remains in white colour.