Abstract:
:Plasmid pUC18rspL is a 3.788-kilobase pair vector, derived from pUC18, pKK232-8 and pHSG664, which identifies promoter-bearing DNA fragments functional in StrA E. coli by activation of a promoterless streptomycin-sensitive gene cartridge (rspL). Expression of the plasmid-borne rspL gene leads to sensitivity dominance and death of the cell. Promoter-bearing DNA fragments can be cloned within a synthetic polylinker containing 12 unique restriction nuclease target sequences. After transformation of StrA E. coli TB1, ampicillin-resistant transformants are replica plated on medium containing ampicillin and streptomycin to identify promoters cloned in their functional orientation. These elements can be sequenced without additional subcloning steps from the M13 universal forward primer hybridization site located 5' of the polylinker in the pUC18 contribution. Transcriptional terminators are cloned 3' of the rspL gene to maintain a balance between transcription and replication when high signal strength promoters such as the E. coli Tac promoter are analyzed. pUC18rspL is used to clone transcriptional promoters from the extreme thermophile T. aquaticus. Promoter signal strength can be estimated by determining the extent of sensitivity dominance conferred to the host.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Vockley JG,Pène JJsubject
Has Abstractpub_date
1990-12-01 00:00:00pages
680, 682-3issue
6eissn
0736-6205issn
1940-9818journal_volume
9pub_type
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