Composite

Part:BBa_K4449010:Design

Designed by: SRIJANI SAHA ,RASHMI RANJAN BEHERA, SATYAM KUMAR SINGH   Group: iGEM22_IISER_Berhampur   (2022-10-05)


Apt4UTI C2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4228
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3485
    Illegal AgeI site found at 4076
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 254


Design Notes

Here Fim H2 will be cloned between the Bam H1 and the Blp 1 downstream of the ATG, thrombin site, and the N terminal 6 X His tag under the control of the T7 promoter. While PCR amplifying the insert a single G base was inserted at the 13 th base of the forward primer sequence (pET15bFimH2 F -BBa_K4449005)while designing primers to maintain the reading frame of the sequence during cloning in expression vector Pet15b. Deleted the ATG start codon from the coding sequence of the insert.

Source

This part was created by cloning FimH2 coding sequence ((BBa_K4449001) in pET15b+ expression vector (Novagen -EMD Millipore)

References

1.Schembri, M.A., Hasman, H. and Klemm, P. (2000). Expression and purification of the mannose recognition domain of the FimH adhesin. FEMS Microbiology Letters, 188(2), pp.147–151.DOI: 10.1111/j.1574-6968.2000.tb09186.x


2.Tao Wang , Wang Yin,Hadi AlShamaileh , Yumei Zhang , Phuong Ha-Lien Tran , Tuong Ngoc-Gia Nguyen , Yong Li , Kuisheng Chen , Miaomiao Sun, Yingchun Hou , Weihong Zhang, Qingxia Zhao, Changying Chen , Pei-Zhuo Zhang, and Wei Duan, 2019 Feb;30(1),A detailed protein-SELEX protocol allowing visual assessments of individual steps for high success rate, DOI: 10.1089/hgtb.2018.237