Here is a depiction of the pSK+ vector: The vector allows for protein expression

ID: 261325 • Letter: H

Question

Here is a depiction of the pSK+ vector: The vector allows for protein expression in E. coli.

Briefly explain the role of the following elements in the context of gene expression and protein production:

LacP:

LacO:

LacZ?:

Briefly explain and indicate what is required for high expression in E. coli in this context. The vector also contains other important elements. Explain their role:

AmpR:

ori:

Please accurately and thoroughly explain the following roles in detail.

Figure Il-1 MCS Xhol669 LacO LacP 5' MCS 234-281) pSK 2964 bp GFP ORF 717 bp Amp AWNI 2811 lac BsrG 1 (5551 ori GFP pGFP 33 kb BsfB 1 (911 grL 3 MCS (1005-1098 Amp AlwNU 1569 Pvu I Aat II (1413) 5 173 amplify GEP gene by PCR (oligos have Xbol Xhol ends Xhol -Restrict plasmid and GFP fragment with Xbol -Remove phosphate groups from pSK with CIP Xhol pSK ligate GFP fragment into pSK plasmid the fragment can ligate in either orientation The AlwNU site is at position 667 in the GFP sequence LacZo/GFP translational fusion AlwNI AlwNI pSK/GFP CORRECT ORIENTATION PSK/GFP INCORRECT ORIENTATION OR AlwNI 1569 AlwNI 1569 Use to transform E. coli

Explanation / Answer

(a) Lac-P it is the promoter region of lac operon. It is the site where RNA polymerase will binds which will result in the expression of the lac genes..

(b) lac-O - it is the operator site which helps in regulation of lac operon. It is the site where the repressor protein binds when thus preventing the binding of RNA polymerase to the promoter site thus keeping the lac operon in closed state when not required.. During activation the repressor protein removes from this site hence polymerase binds to the promoter..

(c) lac-Z- it produces an enzyme called beta galactosidase which results in the breaking of the lactose into glucose and galactose..

(d) AmpR-it is the ampicillin resistance gene which helps the bacteria to gain resistance to the antibiotics ampicillin.. It helps in identifying the cloned bacteria by growing it on the ampicillin resistance plate after transformation..

(e) ori - it is defined as the origin of replication whre the replication process start..!!

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Here is a depiction of the pSK+ vector: The vector allows for protein expression

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