The Development of Expression Vectors for the Production of Useful Proteins

Mathai, Judith Njoki (1989) The Development of Expression Vectors for the Production of Useful Proteins. Masters thesis, Kenyatta University.

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Abstract

A new vector has been constructed by joining the lac Z gene to the EMBL3 vector. The hybrid vector takes advantage of the easy screening provided by lac Z, blue /white colour differentiation of recombinant and non-recombinant phage respectively. The presence of the EMBL3 offers a large capacity for cloned DNA. The strategy employed to construct the vector involved sub-cloning a 12 kb fragment from lambda gtll containing the lac Z gene into a plasmid, pUC18. The recombinant plasmid was amplified and the lac Z-containing fragment recovered and ligated to EMBL3 arms. To achieve this, pUC18 DNA was made and digested with Kpnl and Sall. The digested products were then ligated to lambda gtl1 DNA fragments which had been digested with the same enzymes. This ligation was transformed into the bacterial strain, Escherichia coli JM 83 and the resulting colonies were probed with a radiolabelled 4 kb fragment which contained sequences unique to the region flanking the lac Z gene in lambda gtll. Both blue and white colonies were observed, and the blue colonies were found to hybridize with the probe, and on characterization were found to contain the 12 kb fragment. The 12kb fragment was extracted from the plasmid and treated with Bal 31 enzyme to remove the overhanging ends generated by Kpnl and Sall. The BamHI site within the 12 kb fragment was methylated and ftgmHl linkers added before ligation to EMBL3 arms. The ligation mixture was packaged in Yi1rQ and allowed to infect I.coli Y1090 cells and plated out in the presence of IPTG and X-GAL. Blue plaques were seen and when their DNA was digested with ftgmHl four fragments were observed. Two of these fragments corresponded to the left and right arms of EMBL3,( 20 and 9.6 kb respectively), the other two ,a 6.5 and a 5.0 kb fragment, together corresponded to the BAL31 treated 12 kb fragment containing the lac Z gene. This new vector was designated lambda KET.1 To test the vector, a library of Trypanosoma brucei 221 was made, which contained approximately 6.7x104 phage particles of which 73 % were recombinant. Lambda KET.1 could be improved by eliminating the BamHl internal to the arms to facilitate cloning with BamH1, which would greatly reduce the possibility of cloning parental phage sequences. Further improvement may be achieved by the addition of strong promoters such as the t7 promoter which would have the effect of enhancing the expression of genes of interest.

Item Type:	Thesis (Masters)
Subjects:	Q Science > QK Botany
Divisions:	Africana
Depositing User:	Tim Khabala
Date Deposited:	05 Mar 2018 09:32
Last Modified:	05 Mar 2018 09:32
URI:	http://thesisbank.jhia.ac.ke/id/eprint/3390

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