You are becoming increasingly talented at cloning so you decide to start your ow

Question

You are becoming increasingly talented at cloning so you decide to start your own company.You want to make maize plants that express a commercially viable protein that you can isolate and sell as a product.Maize is a good choice since a lot of biomass can be generated and processed efficiently with current agricultural techniques.You’ve learned that human tears contain an antimicrobial enzyme called lysozyme that destroys bacterial cell walls.You plan to develop an aerosol mist that could help fight nasal infections.You’ve been able to isolate the gene that encodes for human lysozyme (lyz) and now you want to clone the coding region into a plant expression plasmid (pBNJ).You know these commercial expression plasmids have high expression promoters that you are going to need to make lots of lysozyme in maize.Unfortunately, you don’t have a choice for restriction sites and you have to use NcoI to get the lysozyme coding region into pBNJ.You know that the lysozyme coding region has to go into pBNJ in the correct native orientation or functional lysozyme will not be produced.Also, you can’t do a blue/white screen because pBNJ does not have the LacZ gene. The human DNA and pBNJ DNA were both treated with NcoI restriction enzymes. The resulting fragments were allowed to mix under conditions that favored ligation. Competent cells took up the products by transformation. Plasmids were isolated from ampicillin resistant colonies. A subset of the plasmid was digested with restriction enzymes and run on a diagnostic agarose gel. Maps of the original pBNJ DNA and the human DNA are given.

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

You have answers in the paragraph itself. Here, you must use two restriction enzymes for successful insertion of lyz gene into the plasmid. Otherwise, one end will hang on the recombinant vector.

2a) NcoI

2b) Ampicillin resistance (ampr)

2c) 3813+ (320+485+505)= 3813+1310= 5123bp

2d) SacI is absent in pBNJ, so there is no possibility of cloning of the SacI digested Lysozyme gene fragment into the plasmid vector.

Insert Orientation Restriction Enzyme w/in Insert Recombinant Plasmid Position Native KpnI 1841 to 2830 bp (990 bp of lyz gene) Native SacI no recombination occur due to absence of Sac I in plasmid. Non-native KpnI 1841 to 2910 bp (1070 bp of lyz gene and promoter) Non-native SacI no recombination occur due to absence of Sac I in plasmid.

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