Question

Q5. Figure 2 shows the plasmid PGL4.83[hRlucP/Puro]. Based on Figure 2, answer the following: Synthetic poly(A) Amp' Sall 2599 ori Sfil Acc651 Kpnl EcolCRI Sacl Nhel Xhol Bglli D Synthetic poly(A) PGL4.83[hRlucP/Puro) Vector (4815bp) Sfil Hindlll hRlucP Puro SV40 early enhancer/ promoter EcoRI 1033 SV40 late poly(A) signal 5141MA 1431 BamHI Figure 2 (Source: httpwww.biofeng.comzaitīburup GL4.83.html 1)

Answer 1

51. A 1. PolyA sequence can be inserted at oriC and AmpR gene can be added by artifical gene synthesis or using primer with poly A nucleotide at its 5' site.

ii) Artificial gene synthesis we can add any predecide gene sequence while using primer speciifc of OriC or ampR gene with poly A nucleotide at 5' could add poly A nucleotide at end of gene in pcr end product

iv) the gene AB is cloned at hRlucP gene site of plasmid using EcoRI.

So if gene clones in right oreintation than after treating cloned vector with SalI and HindIII, result in two fragments

1st framnet of size = 1000+ (1033-66)= 1967bp

While 2nd band of size= (300+4815-967)=4148bp

After adding both fragments size= 6115 which is size of (palsmid + gene)

If gene clone in wrong orientation so again two fragments form

1st fragments= 300+1033-66=1267bp

2nd fragment size = 1000+4815-967= 4848bp

After adding both fragments size = 6115bp

After treatment with restriction enzyme we run samples on gel and analyze the bad pattern.

v) successfull cloned vector can be quickly analyzied after transformation into E.coli cell. If we grow cell in presence of luciferin, then those cell will emit NO light that cells having succesful cloned gene at hRlucP gene of vector because after gene AB insert at hRlucP gene ,it's become non functional so it cannot synthezied functional lucifarase enzyme and hence not able to convert luciferin to oxyluciferrin and hence no light emission.

While those cells which emit light means luciferase gene is intact and hence that cell doesnot have cloned gene.

Hope it's clear...thanks