Team:MoWestern Davidson/tRNA

Suppressor tRNA Sequence and Design:

From the papers by Magliery, Anderson, and Shultz we obtained the sequence for the tRNAs that they discovered. All of the tRNAs used are based on the same Serine tRNA, the sequence for which we obtained from Dr. Chris Anderson. The only difference between our constructed suppressor tRNAs is a variable 9-bp anticodon loop. To assemble the tRNAs, our team decided to use single stranded overlapping oligos. Since the tRNAs are all the same except for the anticodon loop, oligo assembly allowed us to reuse several of the oligos to make the different tRNAs.

The underlined portion of the oligos are the anti codon loop and its complementary bases on the opposite strand. The different colors represent the different oligo nucleotides. The top strand has 5' and 3' overhangs which are the sticky ends for EcoRI and PstI respectivly. Our team annealed the oligos together and then cloned them into a pSB1A2 vector.

The tRNA sequence is flanked by 5' and 3' context. This context was suggested to us by Dr. Anderson to allow for the processing of the tRNA by the E. coli. The full biobrick prefix and suffix are also included.