1.Pst (part number: BBa_K4252009)
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, PstC, PstA, PstB, and other regulator genes such as phoB, phoR and phoU are used to control the
inoriginic phosphate transport and others like phoA, phoE, phoP to control the other forms of phosphorus.
(1)PstS:
PiBP, a protein which is determined by PstS gene, is also a phosphate-binding protein and discriminates between arsenate and phosphate, which attached
to the outer side of the cell membrane and it could combine with phosphate in periplasmic space and transport it to the membrane (also an ABC-transporter).
(2)PstA and PstC:
PstA and PstC determine pstA and pstC and both of which are hydrophobic protein and they form the transmembrane portion of the Pst system.
(3)PstB:
PstB determine pstB protein that is the catalytic subunit and interact on the cytoplasmic side, which couples the energy of ATP hydrolysis to control the open
and close of phosphate channel by the alpha-helix domains of PstA and PstC. And phosphate molecule can across the channel by the salt bridge composed of Arg and Glu.
(4)Regulator (PhoB, PhoR, PhoU) :
The Pho regulon is controlled by a two-component regulatory system which comprises an inner-membrane histidine kinase sensor protein and a cytoplasmic
transcriptional response regulator. The system in E.coli is named PhoB-PhoR. The PhoB encodes a positive transcriptional activator, phoB, and PhoR encodes a
phosphate-sensory protein, phoR. The PhoB is already a dimer before binding to the DNA, but it has to be phosphorylated in order to become active: it bind a upstream
from genes of the Pho regulon, which is directly for the PstS. The PhoR protein has a dual regulatory role as both an activator and a repressor. Under Pi limitation,
PhoB is activated by PhoR acting as a kinase, but under Pi-replete conditions, PhoB activation is interrupted by PhoR acting as a phosphatase. PhoU is required for
PhoB dephosphorylation under Pi-rich conditions in an unknown way. When phoU is mutated or deleted, PhoR behaves as a constitutive PhoB kinase, leading to high expression
of the Pho regulon genes. PhoU is involved not only in control of the autokinase activity of PhoR, but also in the control of the Pst system to avoid an uncontrolled Pi
uptake that could be toxic for the cell.
2.PPK (part number: BBa_K4252002)
As mentioned above, Escherichia Coli (E. coli) takes up Pi monomers from the environment through the Pi inorganic transport (pit) and Pi-specific transport (pst) systems.
When there is an excess of Pi in the body, Pi will be converted to polyP with the help of poly-phosphate kinase (ppk) and thus stored in the bacteria.
In previous studies, several ppk mutants were identified that can significantly enhance the level of polpP synthesis in vivo. PPK is a tetramer, and these amino acid
mutation sites often occur at the interface between the monomers of the ppk tetramer. Those known ppk mutants can increase polyp accumulation in E. coli and
consequently enhance the ability of taking up Pi from the environment and store it.
Thus, in our design, the mutants identified in the literature were compared across the borad, and a mutation (E240G) that can cause the most significant change in
polyP level in E. coli was selected to enhance the single phosphate storage level.
It is worth mentioning that since ppk is a tetrameric protein, if we transform the plasmid containing mutated ppk genes directly into the bacteria may cause the
wrong arrangement of monomers and prevent ppk from performing its normal function, so we previously used lambda red homologous recombination technology to knock
out the original wild-type ppk gene on the genome before transforming our recombinant plasmids. By this way, we hope to combine this ppk mutant with other components
to enhance the ability of E. coli to transport Pi from the environment.
3.yjbB (part number: BBa_K4252001)
So far, only one type of P exporting protein, YjbB, has been reported in E. coli. The mechanism of phosphorus export by yjbB is still unclear. But based
on experimental phenomena we choose YjbB as the phosphorus output element in our project.
4.T-Switch system (part number: BBa_K4252023)
Our thermal regulation circuit is generally conducted by a thermal-sensitive repressor cI857, a widely used mutant of cI from bacteriophage λ as a thermogenetic tool.
This part consists of two basic domains. A coiled-coil domain, which gathers up with each other to make protein-protein interaction at 30℃, is the functional domain
for its thermal sensitivity. Another domain mainly conduct genetic repressing function of the part when monomer cI857 connect with each other to form dimer.
This repressor can conduct bi-functional regulation if another repressor gene whose expression is regulated by cI857 is added to this system. Here we choose PHLF repressor
to realize it, and finally, our complete genetic circuit is accomplished.
