Protocol for Lock/Key synthesis

1) From the Lock/key generator, the RNA and DNA sequences are obtained. Attaching the prefix and suffix to the DNA sequence will deliver the new biobrick sequence as show below (example with weak RBS).

Lock for weak RBS

5´- GAATTC GCGGCCGC T TCTAGA G GTA GGATTCCTGTGTGA GGAC TTTGGGTAGATCAC TCACACAGGAAACC T ACTAGT A GCGGCCG CTGCAG -3´

3´- CTTAAG CGCCGGCG A AGATCT C CAT CCTAAGGACACACT CCTG AAACCCATCTAGTG AGTGTGTCCTTTGG A TGATCA T CGCCGGC GACGTC- 5´

Key for weak RBS

5´-GAATTC GCGGCCGC T TCTAGA G ACCCAAAGTCC TCACACAGGAAACC TGGTTAATGAAAATTAACTTA GGTTTCCACTGTGA AAAAAGCCGAGTTATTAATCCGGCTT T ACTAGT A GCGGCCG CTGCAG-3´

3´- CTTAAG CGCCGGCG A AGATCT C TGGGTTTCAGG AGTGTGTCCTTTGG ACCAATTACTTTTAATTGAAT CCAAAGGTGACACT TTTTTCGGCTCAATAATTAGGCCGAA A TGATCA T CGCCGGC GACGTC- 5´

2) Double strand DNA synthesis is more expensive than single strand. Therefore in most of the cases the latest is preferred. However, the new biobricks are intended to generate secondary RNA structures which will also be formed in ssDNA (see scheme 1). Therefore the next steps (see also the scheme 2) might be taken into account.

Scheme 1. ssDNA will for a similar secondary structure than the one pretended in the RNA molecule, which generate problems in DNA synthesis

• Attach the prefix (E and X) to the sequence in the position 5’. • Attach only a part of the suffix (only S) to the complementary sequence in the position 5’. • Make small oligonucleotides (half of the original sequence) with overlap regions.

Lock for weak RBS

5´- GAATTC GCG GCC GCT TCT AGA GGT AGG ATT CCT GTG TGA GGA CTT TGG GTA GAT CAC -3´ 5´- GGG ACT AGT AGG TTT CCT GTG TGA GTG ATC TAC CCA AAG TCC TCA C-3´

Key for weak RBS

5´- GAA TTC GCG GCC GCT TCT AGA GAC CCA AAG TCC TCA CAC AGG AAA CCT GGT TAA TGA AAA TTA ACT TA -3´ 5´- GGG ACT AGT AAA GCC GGA TTA ATA ACT CGG CTT TTT TCA CAG TGG AAA CCT AAG TTA ATT TTC ATT AAC CA -3´

• Order the oligonucleotides. • Use the nucleotides as primers in PCR. One reaction showed to be enough. • Purify the PCR products. • Digest with E and S. Do the same with the favorite plasmid backbone. • Ligate the new biobrick and the backbone.

Scheme 2. Graphic representation of the basic steps for synthesis of dsDNA for the new locks and keys