Part:BBa_K4252002
Polyphosphate Kinase E240G (Escherichia coli)
The gene encodes a mutant of polyphosphate kinase(PPK) that has a E-to-G transition at amino acid 240, which increases the efficiency of converting free Pi into polyP in vivo.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 268
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 268
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 268
Illegal BglII site found at 317 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 268
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 268
Illegal AgeI site found at 1671 - 1000COMPATIBLE WITH RFC[1000]
Introduction
For all we know, phosphorus is the key substance that causes eutrophication in water. It will not only lead to the rapid growth of algae in water, but also make the oxygen content in water drop sharply, affecting the survival of other aquatic organisms.
As a consequence, we design this part to construct a PPK mutant element that can significantly enhance the ability of E. coli to convert Pi to polyP for storage in vivo. We improved the existing part BBa_K986000 by setting a mutation site (A719G) on it. Then, we transferred our improved mutated gene into the E. coli MG1655 strain after knocking out the wild-type ppk gene on the genome and induced it by adding 0.6mM IPTG. Expression was then examined by Coomassie Brilliant Blue staining
The ability of our modified mutant to store phosphorus was characterized indirectly by measuring the difference in phosphorus concentration within the bacterial medium at the same growth density of the wild-type and mutant strains. Specific steps can be found in the experiment & results section of our wiki.
Construction
Acquisition and point mutation of wild-type ppk
As shown in the figure, 1 is the wild-type ppk we amplified by PCR from the genome of MG1655 strain, 2 to 4 are overlap PCR results. 2 is fragment A to the left of the mutation site, 3 is fragment B to the right of the mutation site, fragments A and B were used as template and primer for each other, and the final mutant A719G was amplified, i.e. 4.
The length of both wild-type and mutant ppk is 2085 base pairs. Due to an overlapping segment of the primer design, fragment A and fragment B add up to a length greater than 2085.
Screening of mutants
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After performing wild-type ppk knockout on the genome and transformation of the plasmid containing mutant ppk, we picked five monoclonal clones from plates with kanamycin and chloramphenicol dual resistance, extracted the whole genome separately and then amplified them from both ends of the ppk sequence using primers without His Tag to perform preliminary screening for mutants.
The PCR amplification results of genomic DNA from five monoclonal clones are shown in 1 to 5. 1-3 have shallow ppk bands, which might be caused by incomplete wild-type ppk knockout. Hence, we selected 4 and 5 for further validation.
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In this experiment, we inoculated the above two mutants into LB medium and centrifuged (4000 rpm, 10 minutes, at room temperature) until the growth density was approximately the same. Then we washed twice with sterile water and resuspended by adding MOPS minimal medium, continued to incubate (37°C, 225 rpm) and the growth density and phosphorus concentration were measured hourly starting from 4 h after resuspension.
A(710 nm) reflects the phosphorus content in the supernatant, and the higher it is, the more phosphorus is in the liquid (You can also see the standard curve of the concentration of phosphate below here). In the results processing, we subtracted the actual phosphorus concentration from the initial phosphorus concentration and divided it by the growth density, thus reflecting the ability of the bacteria to absorb phosphorus during that period of time.
_standard_curve.png)
From the Figure 3 we can see that mutant 2, although it absorbed phosphorus faster (the supernatant phosphorus concentration after 4 hours of incubation was significantly lower than that of the wild-type after 8 hours of incubation), was not stable and rebounded after 4 h. Mutant 1 also had some ability to absorb phosphorus and was more persistent. So in the subsequent experiments, we first selected mutant 1 for functional verification. The mutant strain were saved as glycerol stock.
Characterization
Validation of expression
Figure 6A, from right to left, shows the induction of the mutant bacterium for 2, 4, and 6 h by adding 0.6 mM IPTG to the mutant broth incubated for a certain time.
Figure 6B, from right, 7th is wild type, 6 to 4 is mutant which has been inducted for 2, 4 and 6 h, 3 to 1 is a ppk overexpressing MG1655 strain (transforming plasmids containing wild-type ppk gene into wild-type E.coli) which has been inducted for 2, 4 and 6 h. The molecular weight of PPK is 80 kDa, and the brightest band of the marker we used is located at 75kDa.
As shown by the staining results, both wild-type PPK and mutant PPK appeared to be well expressed in MG1655 strain, and the expression level increased with induction time.
Validation of function
Before the formal experiments for functional validation began, we performed an initial pre-experiment with a gradient of IPTG concentration, hoping to select a more appropriate induction concentration. In the same way as the experiments mentioned in the second section, we used MOPS minimal medium to resuspend and then measure phosphorus concentration and growth density every hour. We chose 0.2mM, 0.6mM, 1mM and 2mM concentration to compare with the wild-type, respectively.
It is worth mentioning that since we found a large difference in the growth rate between the mutant and wild-type at the same time (see in Figure 8), we chose to plot the growth density versus phosphorus concentration to compare the ability of E. coli to absorb phosphorus under different conditions.
A and B were grown in LB for 12 h. A was the mutant and B was the wild-type;
C and D were grown in LB for 24 h. C was the wild-type and D was the mutant;
The growth rate of the mutant and wild-type is very different at both 12 and 24 hours, and is more pronounced at 24 hours.
From Figure 5 we can observe that the phosphorus concentration in the supernatant of the mutant was significantly lower than that of the wild-type when the IPTG concentration was 0.6 mM, and the growth of bacteria was faster compared with the other groups. Therefore, we selected the IPTG concentration of 0.6 mM for induction in the formal experiment.
In the formal experiments, we inoculated MG1655 wild-type, MG1655 overexpressing ppk and mutant strains into same volume of MOPS minimal medium, and measured the growth density and phosphorus concentration every half hour until the bacteria grew to the plateau stage.
As seen from the experimental results, the bacteria overexpressing ppk grew to an A(600nm) of about 0.6 and then showed a more pronounced phosphorus uptake compared to the wild-type strain, with a significant reduction in the phosphorus concentration in the supernatant.
In contrast, the phosphorus uptake of the mutant was very obvious at the early stage of growth, and the phosphorus concentration in the supernatant at an A(600nm) of about 0.4 was already relatively the same as that of the wild-type at an A(600nm) of about 0.9.
Conclusion and Perspectives
We can see that by modifying the ppk gene, the mutant strain has a much better rapid phosphorus uptake ability than both wild-type and the existing part BBa_K986000. We can expect to combine this modified ppk element with other elements involved in phosphorus transport designed by our team, such as pst (BBa_K4252009) and yjbB (BBa_K4252001), to synthetically enhance phosphorus uptake by E. coli engineered bacteria.
In the subsequent research, this kind of engineering Escherichia Coli can also be fixed to the semi-permeable membrane or some other things to recycle phosphorus in the wastewater and apply in practice. We are eager to contribute in a small way to sustainable development by using this modified engineered bacteria for wastewater treatment.
“Lucid waters and lush mountains are invaluable assets”, we look forward to the day when the ecological environment can be restored as before, and the harmonious development of man and nature can be achieved.
However, at the same time, the phosphorus concentration rebounded in the middle and late stages of mutant growth, indicating that this ppk mutant might have some toxicity, which can also be improved in subsequent experiments. How to combine the characteristics of this component with other components reasonably and give full play to its maximum function is still a subject to be studied.
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] Rudat AK, Pokhrel A, Green TJ, Gray MJ. Mutations in Escherichia coli Polyphosphate Kinase That Lead to Dramatically Increased In Vivo Polyphosphate Levels. J Bacteriol. 2018 Feb 23;200(6):e00697-17.
- [3] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5.
- [4] Zhu Y, Lee SS, Xu W. Crystallization and characterization of polyphosphate kinase from Escherichia coli. Biochem Biophys Res Commun. 2003 Jun 13;305(4):997-1001.
- [5] Itaya K, Ui M. A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta. 1966 Sep;14(3):361-6.
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