Etch-a-Sketch. 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.

Etch-a-Sketch

RBScI434 + LVA Ter

ptrpL

RBST7 P + NLS Ter

Modified pRM

TerpT7

New Simplified First Run Target

mRFP

High copyplasmid

Low copyplasmid

cI + LVARBS

RBSModified LovTap

TerWeak constitutive RBS

trpRRBS

Final Plasmid 2

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

Procedure1. Amplify 8 biobricks and 3 backbonesPlasmid 1: Already assembledPlasmid 2:2. (VioA-E) I712074 + K274002 pSB3T5Plasmid 3:3. Make ptrpL biobrick (K360023)4. (Attach RBS) B0034 + C0052 [CI434R]5. (cI434) K360023 + CI434R pSB2K3 Plasmid 4:6. (Attach RBS) B0034 + C0051 [CIR]7. (pRM + T7) I12040 + I712069 [RT7]8. (RT7 + cI) [RT7] + CIR pSB4A5Finalize:9. Transform with A C K T

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

ptrpL• GTTTCTTCGAATTCGCGGCCGCTTCTAGAGgctgttgacaattaatcatcgaact

agttaactagtacgcaagttcacgTGCTAGCAGCGGCCGCTGCAGGAAGAAAC– 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)

lovTAP• Activated by 470nm light• When active, acts as trpR– Represses ptrpL

• Stays active for about 1 minute

RBS LovTap TerConstitutive

Final Plasmid 1

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