Upload
merry-hillary-bryan
View
220
Download
0
Tags:
Embed Size (px)
Citation preview
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