SBB09Ntbk-Madhvi Venkatesh

Madhvi Venkatesh 14:54, 2 February 2009 (EST)

Today, I got my project ball and I am making a new flagellar display system. I need to consider several possibilities and figure out what would be best to make and the source. The project description if on this page: new flagellar display system

Madhvi Venkatesh 15:40, 9 February 2009 (EST)

Possible places to find EtpA homolog (from CTEC):

APEC 01:k1
CFT073
EHEC 0157:H7
Shigella flexneri

Madhvi Venkatesh 18:21, 10 February 2009 (EST)

Construction files for C and N terminal fliD parts: Note: fliD has an internal BamHI site

Construction of {<fliD!} (N-terminal)
PCR Omv044F/Omv045R on MG1655 genomic DNA   (485bp, gp=A)
PCR Omv045F/Omv044R on MG1655 genomic DNA   (973bp, gp=B)
----
PCR Omv044F/Omv044R on A + B      (1435bp, EcoRI/BamHI)
Sub into pBca9495KC-Bca1144#5     (EcoRI/BamHI, 2974+910, L)
Product is pBca9495KC-M10000      {<fliD!}
----
Omv044F  Forward Bglbricking of {<fliD!}  ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg
Omv044R  Reverse Bglbricking of {<fliD!}  gcagtGGATCCttacttggaattactgttgttttcg
Omv045F  Forward Removing BamHI site in {<fliD!}  cgttaagcggCatccgtgatgcc
Omv045R  Reverse Removing BamHI site in {<fliD!}  ggcatcacggatGccgcttaacg

Construction of {<fliD>} (C-terminal type 1)
PCR Omv044F/Omv045R on MG1655 genomic DNA   (485bp, gp=A)
PCR Omv045F/Omv046R on MG1655 genomic DNA   (970bp, gp=B)
----
PCR Omv044F/Omv046R on A + B      (1432bp, EcoRI/BamHI)
Sub into pBca9495CA-Bca1144#5     (EcoRI/BamHI, 3039+910, L)
Product is pBca9495KC-M10001      {<fliD>}
----
Omv044F  Forward Bglbricking of {<fliD!}  ccagtGAATTCatgAGATCTgcaagtatttcatcgctggg
Omv046R  Reverse Bglbricking of {<fliD>}  gcagtGGATCCcttggaattactgttgttttcg
Omv045F  Forward Removing BamHI site in {<fliD!}  cgttaagcggCatccgtgatgcc
Omv045R  Reverse Removing BamHI site in {<fliD!}  ggcatcacggatGccgcttaacg

Construction of {a~fliD>} (C-terminal type 2)
PCR Omv044F/Omv045R on MG1655 genomic DNA   (492bp, gp=A)
PCR Omv045F/Omv046R on MG1655 genomic DNA   (970bp, gp=B)
----
PCR Omv044F/Omv046R on A + B      (1439bp, EcoRI/BamHI)
Sub into pBca9495CA-Bca1144#5     (EcoRI/BamHI, 3039+910, L)
Product is pBca9495KC-M10002      {a~fliD>}
----
Omv047F  Forward Bglbricking of {a~fliD!}  ccagtGAATTCatgAGATCTtcgcatggcaagtatttcatcgctggg
Omv046R  Reverse Bglbricking of {<fliD>}  gcagtGGATCCcttggaattactgttgttttcg
Omv045F  Forward Removing BamHI site in {<fliD!}  cgttaagcggCatccgtgatgcc
Omv045R  Reverse Removing BamHI site in {<fliD!}  ggcatcacggatGccgcttaacg