dye-linked substrate called X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galacto-pyranoside) into galactose and aninsoluble blue pigment (4-chloro-3-brom-indigo), which allows the bacterial colonies to appear BLUE on anagar plate.Most plasmid vectors, including pUC19, carry a short segment of lacZ that contains coding information for thefirst 146 amino acids of B-galactosidase. The host E. coli strains (ie, DH5¤, which is used in this experiment)contain the lacZAM15 deletion mutation. When the plasmid vector is taken up by the bacteria, a functional B-galactosidase enzyme is produced, which can break down X-gal to create blue colonies.If, however, the plasmid has a cloned gene of interested in the Multiple Cloning Site (PHPU-4), this will disruptlacZ on the plasmid. If there is no lacZ on the plasmid to interact with the mutated lacZ in E. coli, no ß-galactosisdase will be produced. Therefore, if X-gal is in the agar media, there is no enzyme to break it down sothe colonies will appear white:What gene from the lac operon was cloned into the plasmid?What enzyme does that gene encode?What is the lactose analog that we use to mimic lactose?What is the dye-linked suhstrate?What color colonies will be produced if E. coli can produce LacZ when grown with the analog and substrate?Color Fig. 1A below.What color colonies will be produced if an insert is cloned into the lacZ gene on the plasmid?Color Fig. 1B below.Figure 1. Expected Results from Blue/White Screening. Color in the colonies (littlecircles) as you would expect them to look after transformation and screening for blue/White colonies. A, DH5¤ transformed with PUC19;B, DH5¤ transformed with PHPU4. A. Read the following section to review what you learned about plasmids, transformation and antibioticselection (which antibiotic?)Plasmids and Recombinant DNA TechnologyA plasmid is a circular piece of extra-chromosomal DNA (between 2000-10,000 bp) that is normally found inbacteria. In nature, the plasmid will often contain a gene that encodes a protein that allows the bacteria to beresistant to antibiotics, or a protein that allows the bacteria to cause an infection. Plasmids were discovered byresearchers in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest tocreate recombinant DNA. The gene of interest is inserted into the plasmid at restriction endonuclease sites. Theplasmid can then be used as a vector to shuttle DNA to a different host/bacteria.There are many different kinds of plasmids commercially available. All of them contain:1. a selectable marker (i.e., a gene that encodes for antibiotic resistance, color change, etc)2. an origin of replication (which is used by the DNA making machinery in the bacteria as the startingpoint to make a copy of the plasmid)3. a multiple cloning site (MCS). The multiple cloning site has many restriction enzyme sites and is used toinsert the DNA of interest. The multiple cloning site is usually in the middle of a reporter gene like LacZ.A commonly used plasmid that we will use in this lab is pUC19: (New England Biolabs)Ind 91Bam 51HstAPI 179BsmBI 2683Ndel 183Kasl - Narl - Sfol 235Eco01091 2674Aatll - Zral 2617Bell 245Fspl 256Prul 276Pvull 306Hmrl 364Boell 387BeiVI 2542Sspl 2501Acll 2297,lpol - EcoRI 396Banll - Sacl - EcosSKI 402lec651 - Kpnl 408wal - BaoRI - Smal -Хmnl 2294lacza.Begl 2215MCSScal 2177-Hamll 417Nhal 423Prul 2066lecl - Hinell - Sall 429BepMI - BfuAI 433Avall 2059PUC192,686 bpBseDI 1935Sphl 441Acl 1924HindllI 417Fspl 1919Pvull 628Awall 1837NmeI 1822lgll 1813-Bpml 1784Bsrll 1779Bsal 1766Tril 641BsaXI 659BSPQI - Sapl 683Til 781AAIII - Peil 806Doll 908Bse 1753Bmrl 1744oriAhdl 1694BeiVI 1015BseYl 1110AlwNI 1217Bkell 1292A. Which plasmid is used?B. Which antibiotic was used?B. Now read the section on Blue-White Screening to answer the following questions:Blue-White ScreeningThe well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme B-galactosidase. Expression of the lac operon is induced by lactose, and also by a lactose analogue, IPTG(isopropyl B-D-1-thiogalactopyranoside). (To be completely accurate, IPTG binds and inactivates the lac operonrepressor, thereby allowing lac expression). When expressed, the B-galactosidase enzyme can break down a

The lac operon contains a group of related genes (promoter, operator, Lac Z, Lac A, and lac Y) that…

dye-linked substrate called X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galacto-pyranoside) into galactose and an insoluble blue pigment (4-chloro-3-brom-indigo), which allows the bacterial colonies to appear BLUE on an agar plate. Most plasmid vectors, including pUC19, carry a short segment of lacZ that contains coding information for the first 146 amino acids of B-galactosidase. The host E. coli strains (ie, DH5a, which is used in this experiment) contain the lacZAM15 deletion mutation. When the plasmid vector is taken up by the bacteria, a functional ß- galactosidase enzyme is produced, which can break down X-gal to create blue colonies. If, however, the plasmid has a cloned gene of interested in the Multiple Cloning Site (pHPU-4), this will disrupt lacZ on the plasmid. If there is no lacZ on the plasmid to interact with the mutated lacZ in E. coli, no B- galactosisdase will be produced. Therefore, if X-gal is in the agar media, there is no enzyme to break it down so the colonies will appear white: What gene from the lac operon was cloned into the plasmid? What enzyme does that gene encode?. What is the lactose analog that we use to mimic lactose? What is the dye-linked suhstrate? What color colonies will be produced if E. coli can produce LacZ when grown with the analog and substrate? Color Fig. 1A below.

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...