Difference between revisions of "Part:BBa K2152003"
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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 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 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 | + | 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: |
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Revision as of 08:57, 20 October 2021
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).
