The development of expression vectors for the production of useful proteins
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Date
2012-05-18
Authors
Mathai, Judith Njoki
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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 1ac Z gene into a plasmid, pUC18. The recombinant plasmid was amplified and the 1ac Z-containing fragment recovered and ligated to EMBL3 arms.
To achieve this, pUC18 was made and digested with Kpn1 and Sall. The digested products were then ligated to 1ambda gt11 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 radiolabel led 4 kb fragment which contained sequences unique to the region flanking the 1ac Z gene in lambda gt11. 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 wends generated by Kpn1 and Sall. The BamH1 site within the 12kb fragments was methylated and BamH1 linkers added before ligation to EMBL3 arms. The ligation mixture was packaged in vitro and allowed to infect E Coil Y1090 cells and plated out in the presence of IPTG and X-GAL. Blue plaques were seen and when their DNA was digested with BamH1 four fragments were observed. Two of these fragments corresponded to the left and right arms of EMBL3, (20 and 9.6kb respectively), the other two, a 6.5 and a 5.0 kb fragment containing the 1ac Z gene. This new vector was designated 1ambada KET 1
To test the vector, a library of Trypanosoma brucei 221 was made, which contained approximately 6.7x10 4 phage particles of which 73% were recombinant.
Lumbda KET 1 could be improved by eliminating the BamH1 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 in interest.
Description
A thesis submitted in partial fulfillment for the degree of Master of Science, Kenyatta University, Department of 'Botany, Faculty of Science, 1989. The QD 551 .K4M3
Keywords
Proteins