Part:BBa_K2152003
Bacteriophage Phi X 174 lysis gene E(wild type)
Lysis protein E spanning the inner and outer membrane of the bacterial, leading to low local degradations of peptidoglycan, allow the release of cytoplasmic content.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contribution
Group: iGEM Team FZU-China 2021
Author: Jiale Hong
Summary: Successfully demonstrated the lysis function of this gene
Enterobacteria phage phiX174 lysis protein E, also known as ϕX174E, induces host cell lysis. Studies show that ϕX174E can inhibit the activity of the host translocase MraY, which catalyzes the synthesis of lipid I, a necessary step for the host cell wall biosynthesis. We inserted the lysis gene ϕX174E into a plasmid and then transformed the plasmid into the host cell where it was expressed. Protein ϕX174E accumulates inside the cell, and after its concentration reaches a certain threshold, it will induce cell lysis.
We synthesized the ϕX174E gene and ligated it to a pET30a backbone and constructed a plasmid pET30a-ϕX174E. The plasmid was colony PCR-verified and sequencing verified.
We then transformed the plasmid with the correct sequence to BL21(DE3). A single colony was inoculated in a 5 mL fresh LB media, and shaken at 37℃ at 250rpm overnight; then 500 uL of the overnight culture was transferred to a 50 mL fresh media for further growth (OD600 was measured every 30 minutes). When the OD600 reached 1.0, the 50ml cell culture was equally divided into two groups. IPTG was added into one of them (the final concentration of IPTG was 100μM) for lysis protein induction, and the other group does not add IPTG (control). It was observed that the OD600 value of the IPTG group decreased continuously while the OD600 value of the control group continued to increase. It can also be observed by naked eyes that the cell culture in the IPTG group became clear after 2.5 hours of induction, while that in the control group was still cloudy, indicating that bacteria in the IPTG group lysed. The OD600 time courses and pictures of the cell cultures before and after IPTG induction are shown in the figures below:
Fig 1. A time-course result of the lysis induced by ϕX174E. Cells were grown to OD600 = 1 and were induced with 100μM IPTG or an equal amount of water. OD600 values of two groups were measured every 30 minutes.
Fig 2. A photo of cell cultures just before adding water or IPTG.
Fig 3. A photo of cell cultures that were allowed to grow for 2.5 hours after adding water (left) or IPTG (right).
Characterization and improvement contribution made by iGEM23_SDU-CHINA
Group: iGEM 2023 SDU-CHINA
Author: Suiru Lu and Chao Tang
Summary: We designed a auto-lysis system based on this part. The auto-lysis system will express at the late stationary phase and peaks at about 40h.
Usage
We designed a auto-lysis system based on this part. The auto-lysis system will express at the late stationary phase and peaks at about 40h.
Late Stationary Phase Promoter
When the bacteria enter the stationary phase, the physiological state of the bacteria changes significantly. During this phase, many genes will respond to make timely adjustments. This 4 parts BBa_K4583000 (PYU3), BBa_K4583001 (PYU7), BBa_K4583003 (PYU16), and BBa_K4583004 (PYU92) are the promoters of E. coil. Their most notable feature is that they will express in the late stationary phase. Moreover, they are self-inducible promoters, which means that no additional inducers are needed to be added for expression. Exogenous inducers are expensive and need to be added artificially, whereas self-induced promoters are cost-effective and relatively stable. This part is also very safe because it comes from E. coli MG1655, a commonly engineered bacterium.
- Late stationary phase promoter
- Self-inducible promoter without additional inducers
- Biosafety
Characterization of Late Stationary Phase Promoter PYU3 (an example)
You can found the results of other 3 promoters in their own page Our characterization of this part is divided into two main parts.
- First, this promoter was placed upstream of GFP gene, forming a genetic circuit as shown in Fig. 1. This plasmid was transformed into a bacterium containing another plasmid for characterization. Green and red fluorescence were measured at fixed intervals to compare the expression time and intensity of the two.
- Second, this promoter was placed upstream of the BFP gene, forming a genetic circuit as shown in Fig. 3. This plasmid was then transformed into bacteria containing two other plasmids. Green, red and blue fluorescence were measured at fixed time intervals to compare the difference in expression time and intensity between this part and the other two parts.
For plasmid construction methods and other experimental procedures, see the Design page.
1. Protocols
Our experimental conditions for characterizing this part were as follows:
- E. coli MG1655
- 30oC, 48h, under vigorous shaking
- Plasmid Backbone: PACYC
- Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.) and Molecular Devices SpectraMax i3x.
We used GFP (excitation at 485 nm and emission at 528 nm)and BFP (excitation at 400 nm and emission at 450 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-x). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).
2. Characterization using GFP in 2-plasmids bacteria
In this section we used the PACYC plasmid with PYU3 upstream of GFP gene(Fig. 1). We transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) plasmids respectively (6 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 2
As can be seen from the characterization results, with the exception of three combinations (L19-PesaRp-PYU3, L31-PesaRc-PYU3, and L31-PesaRp-PYU3) the expression time of this element is significantly different from that of the other element in terms of time and intensity.
In the case of the combination L19-PesaRwt-PYU3, for example, the expression of the other part peaked at about 12 h, whereas the expression of this part peaked at about 38 h. The expression of the other part peaked at about 12 h, whereas the expression of this part peaked at about 38 h.
3. Characterization using BFP in 3-plasmids bacteria
In this section, we used the PACYC plasmid with PYU3 upstream of the BFP gene (Fig. 3). Based on the results of the last Characterization, we transformed it into L19 and L31 with BBa_K4583009(PesaRwt), BBa_K4583010(PesaRc), BBa_K4583011(PesaRp) and BBa_K4583012(PesaS) plasmid respectively (4 combinations in total) and characterized them using 24-well plates. The characterization results are shown in Fig. 4.
Auto-lysis system
We ligated the E gene to the plasmid backbone we constructed by Gibson's method to obtain PACYC-PYU3-E,PACYC-PYU3-E,PACYC-PYU3-E,PACYC-PYU3-E,PACYC-PYU92-E plasmid(Fig. 3).
Reference
[1] Barrell, B.G., G.M. Air, and C.A. Hutchison, 3rd, Overlapping genes in bacteriophage phiX174. Nature, 1976. 264(5581): p. 34-41.
[2] Gu, F., et al., Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol, 2020. 9(2): p. 209-217.
[3] Talukder, A.A., et al., RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli. FEBS Lett, 1996. 386(2-3): p. 177-80.
[4] Gao, Y., et al., Inducible cell lysis systems in microbial production of bio-based chemicals. Appl Microbiol Biotechnol, 2013.
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