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Competancy And Transformation

Aim: To make E.coli cells competent by chemical treatment using CaCl2 and transform them with pUC18 vector. Introduction: Recombinant DNA technology enables to study a particular gene by introducing them into a host cell (i.e cloning) by using suitable vehicles (vectors). Inserting a foreign nucleic acid segment into a bacteria cell which results in a new genetic trait is called transformation. To transform a bacterial cell, it first needs to be made competent (capable of taking up naked foreign DNA) by physical or chemical treatment. Also, once the DNA is inserted, screening is necessary to isolate transformed cells from untransformed ones. Principle: E.coli, like most other bacteria need to be manipulated so they become competent. Here, competency is induced in E.coli by treating with chloride salts of the metal cation calcium, e.g. CaCl2. The exact mechanism of action is not understood but it is suggested that CaCl2 causes DNA to precipitate outside the cells or perhaps the salt is responsible for change in cell wall integrity. The positive charge allows CaCl 2-DNA complex to bind to negatively charged cell wall of E.coli. Then this is followed by slight heat shock treatment (42 0C) which enables the uptake of DNA by cells. This vector entering the cells may be self ligated into the host. Bacteria that have taken up the plasmid and transformed must be selected, or isolated. Here pUC18 vector is used and the principle of insertional inactivation helps in screening of transformed cells. pUC18 possesses ampicillin resistant gene (first mode of selection) & lacZ’ gene (which codes for α segment of enzyme βgalactosidase). The E.coli is selected such that it contains only β segment of the enzyme. So only when both the segment of enzyme is present in a single host, the enzyme will be active (α complementation). The gene of interest is inserted in the vector within the lacZ’ gene, thus resulting in inactivation of lacZ’. 1. Cells that harbor a normal pUC18 plasmid are ampicillin resistant and possess active β-galactosidase; thus capable of breakdown of X-gal (a lactose analog: 5-bromo-4-chloro-3-indoyl- β-galacto pyranoside) which yields a blue color pigment. 2. Whereas the cells with recombinant plasmid will show ampicillin resistance but lack β-galactosidase activity due to insertional inactivation; thus will not be able to breakdown X-gal resulting in white cell colonies. IPTG (isopropylthio-β-galactoside)is also added to media which acts as an inducer for the enzyme βgalactosidase. Requirements: Reagents1. LB broth 2. 0.1M CaCl2 3. Antibiotic (Ampicillin) sensitive overnight grown culture of E.coli 4. pUC18 vector 5. LB agar plates (Ampicillin + X-gal + IPTG) Plasticwares1. 1.5ml, 2 ml eppendorf tubes 2. Micropipettes 3. Ice box Apparatus-

6. Observe for blue or white colonies.coli host) prevents the indole dye in these cells from leaking into the medium.coli cells were made competent by chemical method.1M CaCl2. Refrigerate it for 1 week after which it can be used for transformation Part 3 – Transformation of competent cells 1.coli culture 1.D at 600nm and adjust it to 0. 4. 2. 5. Result: E. 3. 4. Discussion: Double screening of the transformants was done using antibiotic ampicillin and selectable marker gene lacZ’. Avoid excessive pipetting. . Heating block (set at 420C) 2. 4. Leave it undisturbed in ice box for 30 minutes. Take a loopful of E. Discard the supernatant and add 200µl of 0. 5.1.coli cells Keep it in ice box for 15 to 20 minutes Subject this mixture to heat shock treatment at 420C for 60 seconds Immediately transfer it in ice box Add 800µl of LB broth and incubate it at 370C for 30 minutes Centrifuge it at 10000 rpm for 5 minutes at 40C Discard the supernatant and resuspend the pellet in 100µl of LB broth. 7. 3. Centrifuge at 10000 rpm for 5-7 minutes at 40C. 2. Again take 1ml of this culture in another 50ml LB and incubate overnight at 370C. 6.coli cells competent 1.coli culture from agar slant and inoculate in 100ml LB broth overnight at 370C Take 1ml of this culture and inoculate in 50ml of LB broth overnight at 370C. Streak a loopful of this culture on LB agar plate containing Ampicillin. Check O. The permease enzyme (a part of lac operon found in E. 10µl of pUC18 (1µg/ml) was added to 200µl of prepared competent E. thus medium does not turn blue. X-gal & IPTG and incubate the plates for 24 hours at 370C 9. Centrifuge Methodology: Part 1 – Reviving E. The transformed cells (i.coli culture and centrifuge at 10000 rpm for 7 minutes at 40C. only white colonies were observed and there were no blue colonies which indicated that all cells were successfully cloned.1M CaCl2. 2. Discard the supernatant and resuspend the pellet in 1ml of 0. Incubator (370C) 3. Take 2ml of revived E. On the media plate.4 Part 2 – making the E. The recombinant cells in which lacZ’ is inactive due to insertional inactivation appeared white. 3. White colonies were observed which show that cloning was carried out successfully.e the one in which plasmid has entered – be it ligated into the host or present as a plasmid in cytoplasm) will show ampicillin resistance and grow in media to form blue colonies. 8.