2a) Which restriction enzyme would be used to clone lysozyme coding region? 2b)

Question

2a) Which restriction enzyme would be used to clone lysozyme coding region? 2b) Which insertional marker trait was used to identify colonies with recombinant plasmids?

2c) The size of the recombinant plasmids are___________ bp.

2d) The lysozyme coding region can be inserted into the pBNJ vector in two possible orientations. Determine the base pair position (based on the orientation of the insert) of each of the following sites in the recombinant plasmid maps:

Insert Orientation Restriction Enzyme w/in Insert Recombinant Plasmid Position Native KpnI Answer Native SacI Answer Non-native KpnI Answer Non-native SacI Answer AbrII NCoI KpnI SacI NcoI Human Promoter lyz coding region 505 750 320 485 3150 AbrII Human DNA pBNJ 3813bp NcoI 1030 1840 KpnI 1560 XhoI ori

Explanation / Answer

2a) Based on the given maps for lysozyme coding region and the plant expression plasmid vector, pBNJ, it is clear that, to obtain the full coding region for lysozyme, there is only one option for the restriction site. This site forms the recognition and cleavage site for the restriction enzyme, NcoI. It is the single restriction site spanning both ends of the coding region, and is also present in the cloning vector pBNJ. Since KpnI and SacI are located within the coding region of lysozyme, so restriction cleavage with these enzymes will result in DNA fragments containing partial coding region of lysozyme. Hence, it is important to cleave human DNA only with NcoI to obtain a fully functional lysozyme coding region. Further, for cloning of gene of interest in a plasmid expression vector, it is important to insert the gene downstream of a high expression promoter. As it can be seen in the plasmid map, just downstream of the maize promoter, there is a single restriction site for NcoI, at the bp position 1030. Other restriction sites in the plasmid include those for XhoI and KpnI located far from the promoter, and close to ampicillin resistance gene, at 1560 bp and 1840 bp positions respectively. Since only NcoI restriction site is located downstream of the promoter, gene inserted only in this region would be expressed when the plasmid is transformed in maize cells. So, NcoI restriction enzyme would be used to clone lysozyme coding region.

2b) A recombinant plasmid obtained by ligation of NcoI cut human DNA and pBNJ DNA would contain the lysozyme coding region (the insert) in the region of NcoI restriction site (1030 bp position) in the plasmid. This region is located below the maize promotor region. When the recombinant plasmid is taken up by maize cells, it will result in expression of the human protein, lysozyme. Lysozyme is an antibacterial protein, which hydrolyzes 1,4-betagycosidic linkage between N-acetylmuramic acid and N-acetylglucosamine. These sugars form the backbone of the bacterial cell wall component, the peptidoglycan. By its glycosidic hydrolase activity, lysozyme is able to hydrolyze bacterial cell wall, thus killing bacterial cells. Thus, cells containing recombinant plasmid will be able to grow in the presence of bacteria, or be resistant to bacterial infection. Thus, lysozyme expression was used as an insertional marker to identify maize colonies with recominant plasmid.

(P.S. Since the plasmid contains ampicillin resistance gene, the plasmid transformed maize cells would be able to grow in the presence of ampicillin. But to select for colonies containing recombinant plasmid, lysozyme expression acts as the selective marker)

2c) According to the DNA maps, the plasmid vector has a DNA size of 3813 bp and the lysozyme coding region contains DNA size of (320 + 485 + 505) 1305 bp. When NcoI cuts human DNA, the lysozyme coding region is separated (1305 bp) with NcoI cut-sticky ends. NcoI restriction digestion of pBNJ vector (3813 bp), results in a linearized plasmid with NcoI generated sticky ends. Then, by ligation of both DNA fragments due to the sticky ends leads to formation of recombinant plasmid.

So, the total size of recombinant plasmid would be 3813 + 1305 = 5118 bp or 5.118 kb.

2d) The native orientation of the lysozyme coding region contains the KpnI restriction site at 320 bp position and the SacI restriction site at 485 bp position from the start of the coding regioin. The recombinant plasmid would contain the lysozyme coding region at 1030 bp position (NcoI restriction site location) of the plasmid. So, if the lysozyme gene is inserted in its native orientation, it should contain KpnI site located at 320 bp away or downstream of 1030 bp position, similarly, SacI would be 485 bp downstream of KpnI. However, if the lysozyme gene is inserted in the reverse orientation, SacI would be more closer to 1030 bp position of the plasmid, in other words, it would be about 505 bp downstream of 1030 bp position and KpnI would be located 485 bp downstream of SacI. Based on this understanding, the following positions would be obtained for KpnI and SacI in the recombinant plasmid:

Insert Orientation Restriction enzyme w/in insert Recombinant plasmid position Native KpnI 1350 bp Native SacI 1835 bp Non native KpnI 2020 bp Non native SacI 1535 bp

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