SBB09 Construct45: Difference between revisions
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PCR Omv046F/Omv052R on pBca1256-M10001 ( | PCR Omv046F/Omv052R on pBca1256-M10001 (816bp, gp=C) | ||
PCR Omv052F/Omv044R on pBca1256-M10000 ( | PCR Omv052F/Omv044R on pBca1256-M10000 (674bp, gp=D) | ||
---- | ---- | ||
PCR Omv046F/Omv044R on C + D ( | PCR Omv046F/Omv044R on C + D (1468bp, EcoRI/BamHI) | ||
Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI,2974+910, L) | Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI,2974+910, L) | ||
Product in pBca9495KC-M10059 {<C.fliD.linker1.N.fliD!} | Product in pBca9495KC-M10059 {<C.fliD.linker1.N.fliD!} |
Latest revision as of 11:43, 9 March 2009
Construction of {<N.fliD>} (replaced below)
PCR Omv044F/Omv045R on MG1655 genomic DNA (488bp, gp=A) PCR Omv045F/Omv044R on MG1655 genomic DNA (178bp, small frag cleanup=B) ---- PCR Omv044F/Omv044R on A + B (643bp, EcoRI/BamHI) Sub into pBca9495AC-Bca1144#5 (EcoRI/BamHI, L) Product is pBca9495AC-M10000 {<N.fliD>} ---- Omv044F Forward Bglbricking of {<N.fliD>} ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg Omv044R Reverse Bglbricking of {<N.fliD!} gcagtGGATCCaaaactttgtagcgcatcatcaccgc Omv045F Forward Removing BamHI site in {<N.fliD>} cgttaagcggCatccgtgatgcc Omv045R Reverse Removing BamHI site in {<N.fliD>} ggcatcacggatGccgcttaacg
JCA: I think you want to make this as an N-terminal display system. So, you'll want a {<part!>} version instead of {<part>}. So, I've added a stop codon to Omv044R below:
Construction of {<N.fliD!}
PCR Omv044F/Omv045R on MG1655 genomic DNA (488bp, gp=A) PCR Omv045F/Omv044R on MG1655 genomic DNA (178bp, small frag cleanup=B) ---- PCR Omv044F/Omv044R on A + B (643bp, EcoRI/BamHI) Sub into pBca9495AC-Bca1144#5 (EcoRI/BamHI, L) Product is pBca9495AC-M10000 {<N.fliD!} ---- Omv044F Forward Bglbricking of {<N.fliD>} ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg Omv044R Reverse Bglbricking of {<N.fliD>} gcagtGGATCCttaaaaactttgtagcgcatcatcaccgc Omv045F Forward Removing BamHI site in {<N.fliD>} cgttaagcggCatccgtgatgcc Omv045R Reverse Removing BamHI site in {<N.fliD>} ggcatcacggatGccgcttaacg
Construction of {<C.fliD>}
PCR Omv046F/Omv046R on MG1655 genomic DNA (781, EcoRI/BamHI) Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI, 2974+910, L) Product is pBca9495KC-M10001 {<C.fliD>} ---- Omv046F Forward Bglbricking of {<C.fliD>} ccagtGAATTCatgAGATCTgttgccgcccagaatgcg Omv046R Reverse Bglbricking of {<C.fliD>} gcagtGGATCCcttggaattactgttgttttcg
Bglbricking of {<linker1>}
Wobble Omv047F/Omv047R (115 bp, EcoRI/BamHI) Sub into pBca9495CA-Bca1144#5 (EcoRI/BamHI, 3039+910, L) Product is pBca9495CA-M10002 {<linker1>} -------------------------- Omv047F Forward construction of {<linker1>} basic part ccataGAATTCatgAGATCTtacttcgatgtctggggcgcagggaccacggtcaccgtctcctcaggcggag Omv047R Reverse construction of {<linker1>} basic part ctgatGGATCCtcctcctccagaaccacctccacctgaaccgcctccgcctgaggagacggtgac
Bglbricking of {<linker2>}
Wobble Omv048F/Omv048R (112 bp, EcoRI/BamHI) Sub into pBca9495CA-Bca1144#5 (EcoRI/BamHI, 3039+910, L) Product is pBca9495CA-M10003 {<linker2>} --------------------------- Omv048F Forward construction of {<linker2>} basic part ccataGAATTCatgAGATCTtggacgttcggtggaggcaccaagctggaaatcaaacgtgcggccgctc Omv048R Reverse construction of {<linker2>} basic part ctgatGGATCCacggccttcaatgccgtgatggtgatggtggtgagcggccgcacgtttgatttc
Note: These parts will be assembled using double antibiotic assembly to make a circularly permuted N-terminal displayer part in a KC vector (C.fliD will be assembled with the linker before being assembled with N.fliD). I currently have not included a stop codon at the end of the contruct, but I don't know if I should add one (it didn't seem like it from the circular permutation tutorial).
Alternate Constructions using SOEing Instead of Assembly
PCR Omv046F/Omv051R on pBca1256-M10001 (819bp, gp=A) PCR Omv051F/Omv044R on pBca1256-M10000 (674bp, gp=B) ---- PCR Omv046F/Omv044R on A + B (1471bp, EcoRI/BamHI) Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI,2974+910, L) Product in pBca9495KC-M10058 {<C.fliD.linker1.N.fliD!} ---- Omv046F Forward Bglbricking of {<C.fliD>} ccagtGAATTCatgAGATCTgttgccgcccagaatgcg Omv051R Reverse on {<C.fliD>} for SOEing linker1 ctccgcctgaggagacggtgaccgtggtccctgcgccccagacatcgaagtaCTTGGAATTACTGTTGTTTTCG Omv051F Forward on {<N.fliD!} for SOEing linker1 gtcaccgtctcctcaggcggaggcggttcaggtggaggtggttctggaggaggaGCAAGTATTTCATCGCTGGG Omv044R Reverse Bglbricking of {<N.fliD!} gcagtGGATCCaaaactttgtagcgcatcatcaccgc
PCR Omv046F/Omv052R on pBca1256-M10001 (816bp, gp=C) PCR Omv052F/Omv044R on pBca1256-M10000 (674bp, gp=D) ---- PCR Omv046F/Omv044R on C + D (1468bp, EcoRI/BamHI) Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI,2974+910, L) Product in pBca9495KC-M10059 {<C.fliD.linker1.N.fliD!} ---- Omv046F Forward Bglbricking of {<C.fliD>} ccagtGAATTCatgAGATCTgttgccgcccagaatgcg Omv052R Reverse on {<C.fliD>} for SOEing linker2 gagcggccgcacgtttgatttccagcttggtgcctccaccgaacgtccaCTTGGAATTACTGTTGTTTTCG Omv052F Forward on {<N.fliD!} for SOEing linker2 gaaatcaaacgtgcggccgctcaccaccatcaccatcacggcattgaaggccgtGCAAGTATTTCATCGCTGGG Omv044R Reverse Bglbricking of {<N.fliD!} gcagtGGATCCaaaactttgtagcgcatcatcaccgc