62. alpha complementation (Blue White color selection) 63. What are competent ba
ID: 204463 • Letter: 6
Question
62. alpha complementation (Blue White color selection)
63. What are competent bacterial cells
64. What enzyme does Lac Z code for? (B-galactosidase)
65. What does the Lac Z gene part on the plasmid code for? 6
6. What does the Lac Z gene part on the bacterial chromosome code for?
67. What color colonies are produced by bacteria that did not take up any plasmid, and grown on media containing Amp/X-gal/IPTG?
68. What color colonies are produced when the non-recombinant plasmid is taken up by the bacteria and subsequently grown on LB/AMP/X-gal/IPTG plates?
69. What color colonies are produced when the recombinant plasmid pSKII-/Kan was taken up by bacteria and subsequently grown on LB/Amp/X-gal/IPTG plates?
70. How do you confirm that your colonies have your fragment insert (the gene of interest)?
71. What are the steps that you will do starting from detecting the colony growth on your plate?
76. What is Ethidium Bromide, how does it stain DNA?
78. What size is the resulting recombinant plasmid DNA (pBlueKan)?
79. On which media (+antibiotics) the bacteria containing the recombinant DNA plasmid will grow?
80. Transformation protocol (why's) and transformation efficiency equation.
81. Competent cells
82. Why does the insert go in the plasmid in either orientation?
83. Why did we pick bacteria with potential recombinant DNA that has the gene in either orientations
Explanation / Answer
62) The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. The method is based on the principle of -complementation of the -galactosidase gene. The key to alpha-complementation is the fact that the lac-Z gene product (B-galactosidase) is a tetramer, and each monomer is made of two parts - lacZ-alpha, and lacZ-omega. Researchers determined that if the alpha fragment was deleted, the omega fragment is non-functional; however, alpha fragment functionality can be restored in-trans via plasmid. Hence, the name alpha-complementation.
63) The prerequisite for bacteria to undergo transformation is its ability to take up free, extracellular genetic material. Such bacteria are termed as competent cells. Competent bacterial cells are those which are able to take up DNA usually foreign and usually as a plasmid.
64) lacZ encodes -galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose.
65) The plasmid, pUC18, includes 2 genes. One gene confers resistance to the antibiotic ampicillin. Bacteria that contain this gene are able to grow in the presence of ampicillin, while bacteria that lack this gene are not. The second gene is called the lacZ gene. The lacZ gene codes for –galactosidase, an enzyme normally used by E. coli to digest lactose.
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