Part:BBa_K112805
[T4 holin]
Holins from T4 bacteriophage assemble together to form pores on inner membrane of bacteria allowing lysozyme to reach periplasm and degrade peptidoglycan layer.
Group: (Michigan 2017) Author: (Aaron Renberg) Summary: We improved this part by optimizing the codons for translation in E. coli using IDT’s codon optimization tool, and by eliminating the illegal XbaI site that Imperial College London’s 2011 team found, making it much easier for future iGEM teams to use. The changes we made were T358C, T556C, T563C, and T571C. Additionally, we constructed three different versions (of varying promoter strength) of a temperature controlled kill switch using holin, endolysin and antiholin. Link: https://parts.igem.org/Part:BBa_K2301000
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]
Characterized by CAFA_China 2022
- We constucted a gene circuit include lacZ gene (BBa_I732019) and T4 lysis Device.
- The experimental result shows that the OD600 of recombinant cells with pBAD-lacZ-T4 lysis gene circuit reduced significantly by 2-3 times than non-recombinant cells after induced by different concentrations of arabinose.
Contribution of SCAU-China 2023
What have we done?
To achieve control over toxicant concentration, we introduced components labeled with BBa_K4632016 [1]to create the T4-T4 lysis device. BBa_K4632016 was originally from BBa_K112805. BBa_K112805's codon was optimized for Escherichia coli( E.coli ) expression.
We characterized the component to demonstrate its effectiveness, as detailed in the construction and characterization section.
In our initial validation experiments, we utilized a dual-plasmid system consisting of pBAD24M and pBAD33 to test our device. (Plasmid maps can be found in Figure 1)
Figure 1. Diagram of Quorum sensing-based T4 lysis device verification systems circuit design
We characterized the component to demonstrate its effectiveness, as detailed in the construction and characterization section.
The Quorum sensing-based T4 lysis device verification systems were transformed into Top10 cells and conducted induction experiments using Ara. At regular intervals, the OD600 values and eGFP fluorescence intensity were measured using a microplate reader. A curve with OD600 were plotted as the vertical axis and induction time were plotted as the horizontal axis. If the OD600 value were observed an significantly decreases after the initially increaseing, the expression of the lysis gene could be confrimed. We assessed whether the pathway could limit the maximum expression level based on the trend in eGFP signal changes. If the eGFP expression rate significantly decreases or ceases to increase after the appearance of lysis, the verification is successful. The limitation of the maximum expression level was assessed by the trend of eGFP signal change.
At the 4th hour, the quorum sensing signal reached the threshold, initiating lysis gene expression. The engineered bacteria lysed, resulting in a significant decrease in bacterial density, which stabilized around the 9th hour.
More detail link: https://parts.igem.org/Part:BBa_K4632024| None |