Can someone please help with describing a strategy? 5. You enter a time warp and
ID: 34303 • Letter: C
Question
Can someone please help with describing a strategy?
5. You enter a time warp and find yourself back in 1961 in Jacob and Monod?s lab. Jacob has asked you to help him find the gene for the lac repressor. Fortunately, when you entered the time warp you were carrying a backpack full of the standard tools of a molecular biology lab in 2015, such as restriction enzymes, plasmids, etc. However, you do not have the sequence of the E. coil genome or any of its genes. Describe a strategy to clone the lac repressor gene. If youExplanation / Answer
lac operon is a negative inducible set of gene that regulates the uptake of and metabolism of lactose sugar.it encodes 3 structural genes encoded by lac Z, lac Y and lac A which code for beta galactosidase, lactose permease, galactoside transactelylase, respectively.
since I travel back and have molecular technologies with me I can clone the lac repressor gene into plasmid vectors and screen for the presence of the gene after using the cloned plasmid vector for transforming host cells through electroporation technique.
Starting with the cloning experiment,
Firstly, since I have access to modern molecular tools, I can use pUC19 plasmid vector which is a circular vector and contains Multiple cloning sites(MCS) which is nothing but a polylinker region within the plasmid and it contains restriction sites for various restriction endonuclease enzymes. when treated with any particular endonuclease enzyme, the plasmid vector linearizes and produces sticky ends. We can use 2-3 sets of different restriction enzymes in order to ensure that the same sticky ends are produce even within the gene of interest. for example we can use, EcoR1,BamH1and Pst1 (in separate set of experiment each). after treating the vector with endonucleases.
secondly, extract the DNA containing lac repressor gene. Then, purify the DNA and amplify it with the modern PCR technology i.e. polymerase chain reaction.
Now treat the DNA of interest with the restriction endonucleases. this will create fragments of DNA that can be used for cloning. even Linkers can be used as they provide sticky ends if in case blunt ends maybe produced.
now, mix the fragmented DNA of interest with the cloning vector add DNA ligase enzyme that seals nicks. Only DNA ends complimentary to each other will pair and complete circular vectors will be obtained.
after the cloning has been done, these vectors will be used to transform host E.coli cells. by subjecting cell culture to electroporation, the host cells are made competent and thus, porous cell wall can pick up naked plasmid DNA from the culture medium. this results in transformation of cells.
now for screening for presence of cloned DNA vectors,
Negatively screening for cells that do not contain vector in them, This is done using ampicillin antibiotic, because pUC19 contains ampicillin resistance marker. Thus, cells that do not contain ampR gene will sucuumb the selection process and are killed.
Positive screening for vector containing cells. pUC19 contains restriction sites within the region coding for Lac Z gene. thus, only vectors that have been successfully cloned with the foreign DNA and contain a complete lac Z gene will be functional. Hence only those E.coli cells which contain a complete Lac Z gene within its vector will turn blue during the Blue-White screening, for which the culture is plated onto a medium containing X-gal i.e. 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside which is a galactose containing copound linked to indole ring. X-gal is analogous to lactose which is a natral substarte for Beta galactosidase enzyme (encoded by lac Z). thus, when a complete and functional lac Z is present in the host cell. it breaks down X-gal and releases the 5-bromo-4-chloro-3-hydroxyindole which oxidizes to 5,5'-dibromo-4,4'dichloro-indigo which is blue in color. Thus, after incubation, whichever colonies appear blue in color have been successfully transformed with the recombinant plasmid vector.
Thus, these blue colonies can be picked from the plates and can be further used for finding the gene. now, the colonies can be purified and pure culture of recombinant organisms can be obtained and can then be used to grow in medium containing lactose in absence of glucose. if the cells grow they contain the gene coding of lac repressor. The plasmid DNA can then be extracted from these cells and it can then be used for sequencing using the modern molecular tools.