Part:BBa_K4252023

cI857-R promoter-phlF-pst plasmid

cI857-R promoter-phlF-pst plasmid involved: The temperature-sensitive control element cI857(BBa_K200011) R promoter, phlF repressor(BBa_K1725040) pst(PstS:BBa_K4252005;PstC BBa_K4252006;PstA: BBa_K4252007;PstB: BBa_K4252008;PstSCAB:BBa_K4252009). We use different temperatures to culture the strain. It is estimated that cI857 forms dimer under 30℃ culture conditions to inhibit the R promoter, so that its downstream phlf and pst do not express. The cI857 dimer was depolymerized at 37 ℃, and the inhibition of the R promoter was relieved. The downstream phlf and pst were normally expressed.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1685
  Illegal AgeI site found at 2251
  Illegal AgeI site found at 4166
  Illegal AgeI site found at 5180
  Illegal AgeI site found at 5369
  Illegal AgeI site found at 5935
  Illegal AgeI site found at 7936
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 5000
  Illegal SapI.rc site found at 8952
  

Introduction

The mechanism of temperature regulation of cI857 protein structure has been introduced in the cI857 element section. We use different temperatures to culture the strain. It is estimated that cI857 forms dimer under 30℃ culture conditions to inhibit the R promoter, so that its downstream phlf and mCherry do not express. The cI857 dimer was depolymerized at 37 ℃, and the inhibition of the R promoter was relieved. The downstream phlf and mCherry were normally expressed.
Two plasmids, cI857-PR-phlf-mCherry plasmid and Pphlf-eGFP plasmid, were transferred into BL21 strain. It is estimated that cI857 forms dimer under 30℃ culture conditions to inhibit the R promoter, so that its downstream phlf and mCherry do not express. The phlf promoter in another plasmid can normally turn on the expression of green fluorescent protein eGFP. At this time, green fluorescence can be detected without red fluorescence using the microplate analyzer; The cI857 dimer was depolymerized at 37 ℃, and the inhibition of the R promoter was relieved. The downstream phlf and mCherry were normally expressed, and phlf inhibited the phlf promoter in another plasmid, so that the green fluorescent protein eGFP was not expressed, while mCherry expressed red fluorescence. At this time, red fluorescence should be detected instead of green fluorescence.

cI857(BBa_K200011):

For the temperature control, we use the constitutive expression of cI857, a widely used mutant of cI from bacteriophage λ as a thermogenetic tool. At 30℃ ,The cI857 dimer repressor binds in a cooperative form, by protein-protein interactions of their C-terminal domains that exhibits strong repression on R promoter. At 37℃, the dimer dissociates, So the R promoter works. Through a special coiled-coil domain, we have endowed dimer proteins with molecular characteristics similar to the anneal-melting process of nucleic acid molecules. Under different temperature conditions, protein monomers show specific polymerization or dissociation characteristics, and thus show different activity characteristics. We used the modified cI857 Repressor in the coiled-coil domain to regulate phosphoric acid release and absorption through temperature changes as signals.

R promoter(BBa_K4252025)

Promoter regulated by cI857. When cI857 forms dimer (about 30 ℃), the R promoter will be inhibited, so that the subsequent genes will not be expressed; When its dimer is depolymerized, the inhibition of the R promoter is removed, and then the gene can be normally expressed.

phlF(BBa_K1725040)

A trans acting element that regulates the phlF promoter in another plasmid.

mCherry

Red fluorescent protein

PstS(BBa_K4252005):

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).

PstA(BBa_K4252007) and PstC(BBa_K4252006)

PstA and PstC determine pstA and pstC and both of which are hydrophobic protein and they form the transmembrane portion of the Pst system.

PstB(BBa_K4252008)

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.

Phosphate-specific transportor (Pst)(BBa_K4252009)

The phosphate (Pi)-specific transport system of Escherichia coli (Pst) is a typical ABC transport system composed of four different proteins: PstS, the periplasmic Pi-binding protein; PstC and PstA, integral membrane proteins that mediate the translocation of Pi through the inner membrane and PstB that binds ATP and energizes the transport. The operon that encodes Pst contains five genes in the following order: pstS, pstC, pstA, pstB, and a fifth distal gene, phoU, whose product does not play a role in the transport of Pi. The Pst system encodes an ATPbinding cassette (ABC) transporter involved in the transport of inorganic phosphate (Pi). As a member of the PHO regulon, the Pst operon is induced in response to Pi limitation. In order to allow E. coli to absorb more Pi, we link the Pst (only contains pstS, pstC, pstA, pstB) gene onto the expression vector pET-28a(+) and introduced it into E.coli BL21. Through inducing its expression by IPTG, E. coli BL21 can efficiently absorb Pi when the external Pi isn’t in limitation.

Characterization

Preliminary design and preparation

Aim

To verify that the temperature sensitive regulating element is controlled by temperature.

Method

• Induction:

Plasmid construction：A plasmid containing temperature sensitive regulatory element cI857, R promoter and red fluorescent protein mCherry

• Plasmid construction:

Use snapgene to edit and synthesize cI857 and R promoter in the company, and use homologous recombination to introduce red fluorescent protein into plasmid.

Results

The sequencing result is no problem. cI857, R promoter and mCherry are all in the plasmid.

Function Test

Aim

we cultured the plasmids transferred into cI857,phlF,R promoter and mCherry at 30℃,33℃,37℃ and 42℃,in order to verify that the temperature sensitive regulating element is controlled by temperature.

Method

1. First,transfer the plasmid into BL21 strain.
2. The strain transferred into plasmid was screened for resistance to ensure successful transfer. The method used in this step is monoclonal antibody.
3. Pick out the strains growing on the resistant culture dish and cultivate them at 30 ℃, 33 ℃, 37 ℃ and 42 ℃ for 12h and 15h.
4. Absorbance test of cultured bacteria.(OD600)
5. Crush the cultured bacteria to prevent their continued expression.
6. Fluorescence test of the broken bacteria under the microplate reader.
7. Calculate the ratio of fluorescence intensity to absorbance and record it as the relative fluorescence intensity.
8. Plot with temperature and relative fluorescence intensity.
   

Results

From the fluorescence data corresponding to our temperature gradient, the red fluorescence expression was very weak in the temperature range of 30 ℃ to 34 ℃, indicating that the dimer formed by cI857 inhibited the R promoter at this time, leading to the subsequent weakening of mCherry expression; The strong red fluorescence expression at 37 ℃ to 42 ℃ indicated that cI857 dimer depolymerized and the inhibition of R promoter was relieved, and then mCherry was normally expressed.
In order to determine the better culture time, we selected the values of 12h and 15h for comparison.We found that there was little difference between the absorbance of 12h culture and 15h culture, and even 15h would decrease under some temperatures, so we chose the final culture time of 12h.

Figure 1

Figure 2

In addition, we selected the bacterial solution cultured at 30 ℃, 37 ℃ and 42 ℃ for 12h, and verified that cI857 is controlled by temperature through the relative fluorescence intensity ratio of red fluorescent protein and green fluorescent protein.
From the figure, we can see that the ratio of GFP to mCherry shows a downward trend, and the ratio is very high at 30 ℃, indicating that the R promoter is inhibited at low temperature; The lower ratio at 42 ℃ indicates that the R promoter inhibition is relieved at high temperature. In conclusion, cI857 is regulated by temperature.

Figure 3

In this part, we cultured the plasmids transferred into cI857, phlF, R promoter and mCherry at 30℃ ,33℃ ,37℃ and 42℃ . From the fluorescence data corresponding to our temperature gradient, the red fluorescence expression was very weak in the temperature range of 30 ℃ to 34 ℃, indicating that the dimer formed by cI857 inhibited the R promoter at this time, leading to the subsequent weakening of mCherry expression; The strong red fluorescence expression at 37 ℃ to 42 ℃ indicated that cI857 dimer depolymerized and the inhibition of R promoter was relieved, and then mCherry was normally expressed.
This part has verified that the temperature sensitive control element cI857 is controlled by temperature.

References

Wang X, Han JN, Zhang X, Ma YY, Lin Y, Wang H, Li DJ, Zheng TR, Wu FQ, Ye JW, Chen GQ. Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli. Nat Commun. 2021 Mar 3;12(1):1411. doi: 10.1038/s41467-021-21654-x. PMID: 33658500; PMCID: PMC7930084.