SBB09 Construct47

1) Native C-terminal portion of ompX {N.ompX}
PCR Ovu015/Ovu016 on MG1655 gen.         (322bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5              (EcoRI/BamHI, L) Product is pBca9145-M10012	{N.cpx} --- Ovu015	Construction of OmpX N term part ctagaGAATTCatgAGATCTGGTCAGTCTggtgactacaacaaaaaccag Ovu016	Construction of OmpX N term part gacaaGGATCCgaagcggtaaccaacagaggcaatccaggtgcctac

2) Native N-terminal portion of ompX {C.ompX}
PCR Ovu017/Ovu018 on MG1655 gen.         (196bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5              (EcoRI/BamHI, L) Product is pBca9145-M10013	{C.cpx} --- Ovu017	Construction of OmpX C term part ctagaGAATTCatgAGATCTgcgacttctactgtaactgg Ovu018	Construction of OmpX C term part gacaaGGATCCttaagagcttgcagtacggcttttctcgg

3) SOEing assembly of eCPX
PCR Ovu015/Ovu019 on pBca9145-M10012       (334bp, EcoRI/BamHI = A) PCR Ovu020/Ovu018 on pBca9145-M10013        (194bp, EcoRI/BamHI = B) PCR Ovu015/Ovu018 on A+B                    (505bp, EcoRI/BamHI) Sub into pBca9145-Bca1144#5              (EcoRI/BamHI, L) Product is pBca9495KC-M10014	{<eCPX!} --- Ovu019	SOEing of eCPX gtcgcTTTAGACTGTTTAGATCCgaagcggtaaccaacag Ovu020	SOEing of eCPX GGATCTAAACAGTCTAAAgcgacttctactgtaactgg

Criteria for Linker between N and C terminus:

From the paper, I was able to extract the following information:
 * The linker should be 6 peptides long
 * The first peptide should be Glycine
 * The third and sixth positions were restricted to R/K/S/H/Q/N
 * The substitution A165V resulted in improved display scaffolds
 * The substitution G166S resulted in improved display scaffolds
 * The display enhancing substitutions A165V and G166S are located immediately upstream of the native C-terminus of OmpX
 * The remaining positions (2 and 4) should be randomized

keeping all this in mind I thought the following linker would be best suited: GSKNVS

which has a nucleotide sequence of: GGTTCTAAAAATGTTTCT

New N junction gttggttaccgcttc

New C junction aaaaaaattgcatgtctttc

Forward Oligo:

Linker.C junction GGTTCTAAAAATGTTTCTaaaaaaattgcatgtctttc

Reverse Oligo:

N junction.Linker gttggttaccgcttcGGTTCTAAAAATGTTTCT

Reverse Complement: AGAAACATTTTTAGAACCgaagcggtaaccaac

Note: VU021F had to be used instead of re-using VU017 because VU017 was used to construct {C.OmpX>} through EIPCR