1. Etch-a-Sketch

2. RBS cI434 + LVA Ter ptrpL RBS T7 P + NLS Ter Modified pRM Ter pT7 New Simplified First Run Target mRFP High copy plasmid Low copy plasmid cI + LVA RBS Modified LovTap Ter Weak constitutive RBS trpR RBS

3. Final Plasmid 2

4. Biobricks Complete LovTAP Plasmid—K360127—Ordered, 887bp – pSB1C3, 2070bp, Chl T7 Promoter—I712074—Plate 1—6N, 46bp – pSB1AK8, 3426bp, Amp, Kan VioA-E—K274002—Plate 3—12B, 7345bp – pSB1T3, 2463bp, Tet ptrpL—K360023—get synthesized, 49bp + prefix/suffix = 108bp – Will put into pSB1C3, 2070bp, Chl cI434 + LVA—C0052—Plate 1—4G, 669bp – pSB1A2, 2079bp, Amp cI + LVA—C0051—Plate 1—4E, 750bp – pSB1A2, 2079bp, Amp Modified pRM—I12040—Plate 1—20D, 91bp – pSB2K3, 4425bp, Kan RBS+T7 P+NLS—I712069—Plate 2—13K, 2678bp – pSB1AK3, 3189bp, Amp, Kan (Strong) RBS—B0034—Plate 1—2M, 12bp – pSB1A2, 2079bp, Amp Part—biobrick#—Plate—Well, length – Plasmid backbone, length (of plasmid), resistance

5. Procedure 1.Amplify 8 biobricks and 3 backbones Plasmid 1: Already assembled Plasmid 2: 1.(VioA-E) I712074 + K274002  pSB3T5 Plasmid 3: 1.Make ptrpL biobrick (K360023) 2.(Attach RBS) B0034 + C0052 [CI434R] 3.(cI434) K360023 + CI434R  pSB2K3 Plasmid 4: 1.(Attach RBS) B0034 + C0051 [CIR] 2.(pRM + T7) I12040 + I712069 [RT7] 3.(RT7 + cI) [RT7] + CIR  pSB4A5 Finalize: 1.Transform with A C K T

6. Plasmid Backbones pSB3T5—Plate 1—7C, 3252bp – Medium copy-p15A – Tetracyline resistance pSB2K3—Plate 1—5C, 4425bp – Low copy-F’/P1, High copy inducible with IPTG – Kanamycin resistance pSB4A5—Plate 1—1I, 3395bp – Low copy-pSC101 – Ampicillin resistance

7. ptrpL GTTTCTTCGAATTCGCGGCCGCTTCTAGAGgctgttgacaattaatcat cgaactagttaactagtacgcaagttcacgTGCTAGCAGCGGCCGCTGCA GGAAGAAAC – 108bp – Uppercase letters are modified biobrick prefix/suffix – Bold is trpR binding site (CTAG sequences in particular) – Underlined are SpeI restriction sites which kill RFC10 Switch SpeI to NheI in biobrick suffix (NheI can still ligate to XbaI) Idea from UNAM 2010 Ligate into linearized backbone pSB1C3 (2070bp) – EcoRI + PstI – Transform onto chloramphenicol plates Submit to library (need special permission)

8. lovTAP Activated by 470nm light When active, acts as trpR – Represses ptrpL Stays active for about 1 minute RBS LovTap Ter Constitutive

9. Final Plasmid 1

10. Plasmid 1 Biobricks (high copy) Weak Constitutive, RBS, modified LovTAP, Ter – Ordered 6/25, (887bp) – pSB1C3, 2070bp, Chl Ter-B0015—Plate 1—23L, 129 bp – pSB1AK3, 3189bp, amp & kan pT7 - I719005, Plate 1- 15N, 23bp – pSB1A2, 2079bp, Amp RBS-B0034—Plate 1—2M, 12bp – pSB1A2,2079bp, Amp mRFP- BBa_E1010, Plate 1- 18F, 681bp – pSB2K3, 4425bp, kan Ter- B1006,Plate 1- 23L,129bp – pSB1AK3, 3189bp, amp & kan Part—biobrick#—Plate—Well, length – Plasmid backbone, length (of plasmid), resistance