SBB09 Construct45

Construction of {} (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  {}

Omv044F Forward Bglbricking of {}  ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg Omv044R Reverse Bglbricking of {}  cgttaagcggCatccgtgatgcc Omv045R Reverse Removing BamHI site in {}  ggcatcacggatGccgcttaacg

JCA: I think you want to make this as an N-terminal display system. So, you'll want a {} version instead of { }. So, I've added a stop codon to Omv044R below:

Construction of {}  ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg Omv044R Reverse Bglbricking of {}  gcagtGGATCCttaaaaactttgtagcgcatcatcaccgc Omv045F Forward Removing BamHI site in {}  cgttaagcggCatccgtgatgcc Omv045R Reverse Removing BamHI site in {}  ggcatcacggatGccgcttaacg

Construction of {} PCR Omv046F/Omv046R on MG1655 genomic DNA (781, EcoRI/BamHI) Sub into pBca9495KC-Bca1144#5 (EcoRI/BamHI, 2974+910, L) Product is pBca9495KC-M10001  {}

Omv046F Forward Bglbricking of {}  ccagtGAATTCatgAGATCTgttgccgcccagaatgcg Omv046R Reverse Bglbricking of {}  gcagtGGATCCcttggaattactgttgttttcg

Bglbricking of { } Wobble Omv047F/Omv047R        (115 bp, EcoRI/BamHI) Sub into pBca9495CA-Bca1144#5  (EcoRI/BamHI, 3039+910, L) Product is pBca9495CA-M10002     { } -- Omv047F  Forward construction of { } basic part ccataGAATTCatgAGATCTtacttcgatgtctggggcgcagggaccacggtcaccgtctcctcaggcggag Omv047R  Reverse construction of { } basic part ctgatGGATCCtcctcctccagaaccacctccacctgaaccgcctccgcctgaggagacggtgac

Bglbricking of { } Wobble Omv048F/Omv048R        (112 bp, EcoRI/BamHI) Sub into pBca9495CA-Bca1144#5 (EcoRI/BamHI, 3039+910, L) Product is pBca9495CA-M10003     { } --- Omv048F  Forward construction of { } basic part ccataGAATTCatgAGATCTtggacgttcggtggaggcaccaagctggaaatcaaacgtgcggccgctc Omv048R  Reverse construction of { } 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 {} 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