Coding

Part:BBa_K4252009:Experience

Designed by: Yuhan Wu   Group: iGEM22_HNU_China   (2022-09-26)
Revision as of 08:13, 11 October 2022 by WuYuhan (Talk | contribs) (→‎Obtain PstSCAB Part)


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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.

Characterization

Expression Test

Aim

To test whether the gene we introduced into the plasmid can be correctly expressed.

Method

  • Induction:

Set two variables：the concentration of IPTG and the induced time.IPTG(mM):0,1,2,4; Induced Time(h):2,4,6.
The constructed expression E.coli strain was inoculated in 5mL LB liquid medium at a ratio of 1:100, and was cultured to OD=0.6. The corresponding IPTG was then added according to the gradient and induce the corresponding time.

  • Sample lysis:

After induction, 4000 rpm, centrifugate for 15 min. Pour out the supernatant, suspend each tube with 5mL cold PBS, and crush it with ultrasonic crusher for 5-10min.

  • Incubation :

After crushing, take 1mL of crushed bacterial solution from each tube, add 50ul nickel medium, and incubate it for 2h at 4 degrees with rotation.

  • Protein denaturation:

Add 7.5uL SDS-PAGE Loading in each tube，boil at 100 ℃ for 10min.

  • Loading and running the gel:

7.5uL sample was loaded onto SDS-PAGE gel.Run the 5% stacking gel for 30min at 80V and run the separating gel for 1 h at 120 V.

  • Wetern Blot:

Then electrophoresed and transferred to nitrocellulose membranes (0.2 µm), which were blocked with 5% non-fat blocking grade milk and incubated with the following primary antibodies overnight at 4 ℃: anti-His (1:2000). On the following day, wash the membrane in three washes of TBST, 10 min each. Then the membranes were incubated with the appropriate secondary antibody (1:10000) at room temperature for 1 h. Immunoblots were then visualized with chemiluminescence reagent kit.

Results

Figure 5: Results of SDS-PAGE electrophoresis.

His Tag is attached to the C segment of PstB protein, which is 257 AA and weighs 28 kD. There was no significant difference in expression between the experimental group and the control group after 2h and 4h induction. After 6 h induction, significant difference can be seen, and the experimental groups with the inducer IPTG concentration greater than 1 mM all have significant expression.

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