Coding

Part:BBa_K4252009:Experience

Designed by: Yuhan Wu   Group: iGEM22_HNU_China   (2022-09-26)
Revision as of 07:47, 11 October 2022 by WuYuhan (Talk | contribs) (→‎Construction)


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Applications of BBa_K4252009

Introduction

Phosphorous, in the form of phosphate, is a key element in the nutrition of all living beings. In nature, it is present in the form of phosphate salts, organophosphates, and phosphonates. Echerichia coli transport inorganic phosphate by two different routes. The first one is a low-affinity transport system, Pit system (phosphate inorganic transport system), which is expressed constitutively and is dependent on the proton motive force, catalyzes a rapid transport process between both sides of phosphate pools. The other way is called Pst system (phosphate-specific transport system). Research has revealed that the high-affinity Pst system (PstSCAB) is induced at low external Pi concentrations by the pho regulon and is an ABC (ATP-binding cassette) transporter, which means Pst system will still work when the concentration of external Pi is lower than 20 microM instead of Pit system.Whereas the high affinity of Pst and its character of inducible expression, our team decide to transfer pstSCAB into Escherichia coli. The Pst system consists of four components, in order of PstS (BBa_K4252005) ,PstC(BBa_K4252006) ,PstA(BBa_K4252007),PstB(BBa_K4252008).

Construction

Obtain PstSCAB Part

  • The PstSCAB part was extracted from the MG1655 genome using NEST PCR (To enhance the specificity）

outer primer F：5’-ACAATGCTTTATGAATCCTCCC-3’
outer primer R：5’-GACTGTCCATAACGCACTCC-3’
Intra primer F: 5’-ATGAAAGTTATGCGTACCACCG-3’
Intra primer R: 5’-TCAACCGTAACGACCGG-3’

  • His Tag was added to the 5’end of the PstB for the detection of the expressing protein

F primer: 5’-ATGAAAGTTATGCGTACCACCGT-3’
R primer: 5’-TCAATGGTGATGGTGATGATGACCGTAACGACCGGTGATG-3’

  • Adding homologous arms to both ends of the PstSCAB part

F primer: 5’-ctttaagaaggagatataccATGAAAGTTATGCGTACCACCGT-3’
R primer:5’-ttgttagcagccggatctcaTCAATGGTGATGGTGATGATGAC-3’

Figure 1: After adding His Tag and homologous arms, the pstSCAB gene is checked with agarose electrophoresis gel and it is 3991bp long.

Construct Recombinant Strains

Then the PCR product was inserted to the expression plasmid pET-28a (+) using methods of homologous recombination. After amplification in DH5α strain, the recombinant plasmids were transformed into the BL21 strain for expression.

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