Part:BBa_K4252009:Experience

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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 2: 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.

Function Test

Aim

To confirm the function of engineering bacteria, we designed a series of experiments and finally we concluded that the engineering bacteria which was transferred an additional Pst gene could absorb the phosphate more efficiently.

Method

1. First prepare the MOPS medium.
2. Inoculate the engineering bacterium into MOPS medium at a ratio of 50 to 1, and add kanamycin resistance at a ratio of 1000 to 1. Grow the engineering bacterium in the condition of 37℃, 220rmp.
3. Take out 4mL of bacterial solution every 0.5h, 2mL of which is used to detect bacterial turbidity, and the remaining 2mL of bacterial solution is centrifuged, taken from the supernatant, and diluted ten times for subsequent detection of phosphorus concentration.
4. When the OD600 of the bacterial solution reaches 0.3~0.5, divide the bacterial solution into two parts, one of which is added with IPTG with a final concentration of 2mM, and the other is not treated.
5. Repeat step 3 until the bacterium no longer grows and the phosphorus concentration value detected subsequently does not change.
6. Prepare 5% potassium peroxydisulfate solution, 20g/L ascorbic acid solution and 26g/L molybdate solution. (The methods to prepare them are as follows)
7. Take 2mL 10 times diluted supernatant ( mentioned in step 3) into a 50mL volumetric flask, then add 2mL 26g/L molybdate solutionand wait for half a minute. Then add 3mL 20g/L ascorbic acid solution and set the volume to the scale with water.
8. Shake volumetric flask and set aside for 15 minutes.
9. Prepare a reference solution in the same way. (Just replace the solution in step 2 with deionized water)
10. Use the reference solution and sample solution to measure the light absorption at a wavelength of 710 nm
    

Results

We have determined the cell concentration (CE), which was represented by the absorbance at the wavelength of 600nm (A(600nm)), and phosphate concentration (Cpi) in the supernatant after centrifugation, which was measured by ammonium molybdate spectrophotometry and expressed by the absorbance at the wavelength of 710nm (A(710 nm)).
Here, the content of phosphoric acid, which is absorbed by the engineering bacteria, was expressed by the reduction of phosphate concentration (ΔCpi). In the meantime, due to the different experimental condition, it is impossible to ensure that each group of test samples will have the same bacterial concentration, so we divided ΔCpi by CE to show the influence of engineered bacteria on external phosphorus concentration.

Figure 3

Figure 4

In this part, we induced the overexpression of Pst system at the point that at the beginning of its logarithmic growth phase and the phosphate absorption capacity is lowest. We have found that the sample which is induced by IPTG grows slower than uninduced one and seems harder to reach the stationary stage (Figure 3).Based on this, we speculated that because the overexpression of Pst system will take up energy and nutrition that is also needed for growth and leads to a low cell concentration of engineering bacteria. After adding 2mM IPTG for induction, bacteria per unit of turbidity had a better ability to absorb inorganic phosphorus than the control group without induction. What’s more, the difference between experimental group and control group is becoming more and more apparent (Figure 4), and eventually stopped increasing then reached a balance after 8 hours induction.
This part has verified that the Pst system has successfully been transferred and could be induced and expressed successfully, and finally enhance the phosphate absorption capacity of the engineered bacteria.

References

[1] Hirota R, Kuroda A, Kato J, Ohtake H. Bacterial phosphate metabolism and its application to phosphorus recovery and industrial bioprocesses. J Biosci Bioeng. 2010 May;109(5):423-32.
[2] Harris RM, Webb DC, Howitt SM, Cox GB. Characterization of PitA and PitB from Escherichia coli. J Bacteriol. 2001 Sep;183(17):5008-14.
[3] Rao NN, Torriani A. Molecular aspects of phosphate transport in Escherichia coli. Mol Microbiol. 1990 Jul;4(7):1083-90.

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