Cloning of minor E.coli tRNA's

Duncan Clark Duncan at genesys.demon.co.uk
Fri Jan 27 09:28:20 EST 1995


Hi Folks,

A few months ago there was a discussion about protein expression in 
E.coli possibly linked to minor tRNA's usage. Well I finally got 
around to PCRing and cloning the minor tRNA's Gly1 (GGG), Arg4 
(AGA), Pro2 (CCC) and Arg5 (AGG). Arg3 and Pro1 are part done. They 
are all cloned separately under their own promoters in the TA cloning 
kit with RE sites built in at either end. The intention is to put them
one after t'other, in the pSL1180 super polylinker vector then move 
them as one to a colE1 compatible vector ie pACYC or something based 
on an R6K replicon. Before I go to the hassle of subcloning them I 
want to know whether functional tRNA is being produced. I do not do 
any sequencing here although that is probably best, but is there any 
alternative ie an easy functional assay etc. As the PCR fragments 
were a maximum of 250bp I do not expect any errors (no flames 
please). 

I am prepared to make these freely available but do not want 
the hassle of distribution. It would make more sense to put them in 
the ATCC or similar ( E.coli Stock Genetic Centre??) but they need 
checking for functionality. Any offers on sequencing gratefully 
received.     

Duncan

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