Part:BBa_K5343011
J23113-RBS(BBa_B0033)-RFP
Usage and Biology
The role played by the ftsz gene in cell division:
Cell division in E. coli is highly regulated both spatially and temporally. more than 20 proteins were recruited to form the E. coli divisome using FtsZ as a scaffold, including early divisional proteins consisting of the highly conserved FtsZ and the membrane anchor FtsA, as well as the less conserved ZipA, EzrA and Zaps, which are responsible for assembling the Z-loop. the Z-loop is assembled with approximately 20% of the cell cycle lag time, followed by the recruitment of late dividing proteins, including the transpeptidases FtsI and FtsW, which are essential for septal wall synthesis.
Construction of polyploids by controlling the intensity of ftsz gene expression:
ftsZ is a major gene that controls cell division, recruiting other division proteins in the form of a ‘Z-ring’ to precisely regulate the process of cell division. ftsZ is weakly expressed below its threshold to prevent cell division, thus preventing a new round of chromosome replication while still allowing the ongoing replication process to complete. Such cells grow in filaments and contain increased numbers of incorrectly segregated chromosomes, which are distributed intracellularly in filamentous extensions in filamentous cells. However, prolonged inhibition of division leads to the eventual lysis of filamentous cells due to cell cycle arrest. In contrast, strong expression of ftsZ allows cells to survive normal division and chromosome segregation. Therefore, it may be possible to construct polyploid E. coli by regulating the expression level of ftsZ to appropriate concentrations.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 680
Illegal SapI.rc site found at 62
Characterization
We characterised this element by the intensity of expression of the reporter gene rfp： Characterisation plasmids pCL-100RFP, pCL-110RFP, pCL-116RFP,pCL-109RFP, pCL-113RFP and pCL-103RFP were constructed by fusing the reporter genes rfp and J23100/J23110/J23116-B0034 or J23103/J23113/J23109-B0033 to plasmid pCL1920 by seamless DNA cloning
Protocal
Our experimental conditions for characterizing this part were as follows:
- Plasmid Backbone: pCL1920
- Chassis cell ：E. coli DGF-298
- Medium: 1.5 mL of liquid LB medium
- Condition 37°C, 24 h, under vigorous shaking
- Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.)
Fluorescence intensity was detected using an enzyme marker at an excitation wavelength of 590 nm and an emission wavelength of 645 nm, and fluorescence intensity values (a.u.) were obtained by calculating the fluorescence intensity to OD600 ratio.
Result：
Characterisation results of promoter and RBS combinations of different strengths：
We selected B1006 terminator with 99% transcription termination efficiency and various strength promoters and RBSs from the iGEM website. the strengths of J23103/J23113/J23109-RBS33 and J23116/J23110/J23100-RBS34 expression elements were shown by the characterisation results to be 32.4 a.u., 40.7 a. u., 51.4 a.u., 652.8 a.u., 1014.9 a.u., and 2148.2 a.u.
Construction of polyploids by regulating the intensity of ftsz gene expression：
The aforementioned expression elements controlled the intensity of ftsZ in strain DGF-298-103Z、DGF-298-113Z、DGF-298-109Z、DGF-298-116Z、DGF-298-110Z、DGF-298-100Z, respectively. As the intensity of ftsZ expression increased from 669.4 a.u. in strain DGF-298-116Z to 2047.0 a.u. in strain DGF-298-100Z, the shape of E. coli changed from filamentous to elliptical. For example,in the present work, the polyploid E. coli DGF-298-103Z designed by SDU-CHINA had significant morphological differences from the starting strain DGF-298, thus verifying that controlling the ftsz intensity can introduce polyploidy on the basis of prokaryotes
After observing it using an electron microscope, we statistically analysed the DGF-298-103Z and found that it has a much larger volume and surface area, confirming our previous conjectures.
Reference：
[1] Descoteaux A, Drapeau GR. Regulation of cell division in Escherichia coli K-12: probable interactions among proteins FtsQ, FtsA, and FtsZ. J Bacteriol. 1987 May;169(5):1938-42. doi: 10.1128/jb.169.5.1938-1942.1987. PMID: 3032902; PMCID: PMC212053.
[2] Belhumeur P, Drapeau GR. Regulation of cell division in Escherichia coli: properties of new ftsZ mutants. Mol Gen Genet. 1984;197(2):254-60. doi: 10.1007/BF00330971. PMID: 6097794.
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