Composite

Part:BBa_K4449011:Design

Designed by: SRIJANI SAHA , RASHMI RANJAN BEHERA , SATYAM KUMAR SINGH   Group: iGEM22_IISER_Berhampur   (2022-10-06)


Apt4UTI C3


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4754
    Illegal EcoRI site found at 5482
    Illegal XbaI site found at 5292
    Illegal SpeI site found at 3729
    Illegal SpeI site found at 4107
    Illegal SpeI site found at 5250
    Illegal PstI site found at 3880
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4754
    Illegal EcoRI site found at 5482
    Illegal NheI site found at 10443
    Illegal SpeI site found at 3729
    Illegal SpeI site found at 4107
    Illegal SpeI site found at 5250
    Illegal PstI site found at 3880
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4754
    Illegal EcoRI site found at 5482
    Illegal BamHI site found at 5111
    Illegal XhoI site found at 10404
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4754
    Illegal EcoRI site found at 5482
    Illegal XbaI site found at 5292
    Illegal SpeI site found at 3729
    Illegal SpeI site found at 4107
    Illegal SpeI site found at 5250
    Illegal PstI site found at 3880
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4754
    Illegal EcoRI site found at 5482
    Illegal XbaI site found at 5292
    Illegal SpeI site found at 3729
    Illegal SpeI site found at 4107
    Illegal SpeI site found at 5250
    Illegal PstI site found at 3880
    Illegal NgoMIV site found at 126
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Here since Bap like Bhp will be cloned between the Nco1 and Xho 1, to get rid of the N terminal His tag, thrombin site, and the T7 tag and retain the C terminal His tag with the expressed Bhp protein, since the C terminal His tag allows proper protein folding and this will ensure reduced interference of our aptamer binding to the thrombin site and T7 tag while performing binding assays.

To maintain the reading frame of the expressed sequence during cloning in expression vector Pet28b+ two G bases were inserted at the 10th and 11th position of the designed primer sequence to maintain the reading frame of the expressed sequence during cloning in expression vector pET28b+.

Source

This construct is obtained by cloning the gene encoding Staphylococcus epidermidis Bap like Bhp ( coding sequence obtained from European Nucleotide Archive browser) in pET28b+ expression vector (EMD Biosciences) between the Nco1 and Xho1 restriction sites.

References


1. Amirmorteza ,EbrahimzadehNamvar, Sara Bastarahang, Niloufar Abbasi, Ghazaleh Sheikhi, Ghehi ,Sara, Farhadbakhtiarian, Parastoo Arezi,Mahsa Hosseini,Sholeh Zaeemi Baravati, Zahra Jokar,Sara Ganji Chermahin ,Clinical characteristics of Staphylococcus epidermidis: a systematic review ,2014 Sep 30, GMS Hygiene and Infection Control 2014, Vol. 9(3),
2.Shankar Upadhyayula, Mamatha Kambalapalli, and Basim . Asmar, Staphylococcus epidermidis Urinary Tract Infection in an Infants;24 July 2012,Case Reports in Infectious Disease,Volume 2012, Article ID 983153.
3.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.
4.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,Human Gene therapy