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Cell Lysis Experiments
Introduction
Cell Lysis is initiated upon recognition of the heat signal by the Heat Sensor. Development and test of the heat sensor is shown on the Heat Sensor Experiments page. Here we show the induction of cell lysis ans subsequent gfp release to the supernatant with the heat sensor.
![TlpA heat sensor induces protein E](https://static.igem.org/mediawiki/2017/d/d7/T--ETH_Zurich--TlpA_Heat_Sensor_general.png)
For more details about the mechanism, go to Cell Lysis.
Phase I: Initial System Design
Protein E mediated cell lysis needs a single gene calles protein E to get activated. In the initial design we placed protein E under an inducible promotor called PLux. A ribosome binding site with large translation initiatio rate was calculated with the Salis Lab RBS calculator and placed in front of the protein E coding sequence. The promotor together with the RBS was ordered as Oligonucleotide and combined with a PCR amplified protein E coding sequence from a plasmid provided by Dr. Irene Weber in a Ligase Cycling Reaction.
![FIXME](https://static.igem.org/mediawiki/2017/f/f5/T--ETH_Zurich--Cell_Lysis_Phase_I.png)
No transformants could be obtained probably due to a high leakiness of the PLux, that lead to enough expression of protein E to lyse all transformants.
We knew now that the protein E must be regulated by a very tight promotor. We used this knowledge to engineer the TlpA heat sensor to a low base level expression of the regulated gene.
Phase IV - A: Combined Function test with reporter gene
A sequence of experiments was performed to find optimal induction times and experimental setup. E. Coli Top10 chemical competent cells were used because of their similarity to Nissle and transformation potency.
Protein E RBS library creation
A ribosome binding site library was created to find variants translating less protein E RNA. The Red Libs algorithm was used and set to calculate degenerate sequences that produce 144 variants. The variants should all have a rather low expression rate to reduce the cytoplasmic amount protein E, produced by leakiness of the promotor. Degenerate primers were ordered at Microsynth and the library was created with a PCR amplification and subsequent Gibson assembly and transformation. The plasmid was designed in a way that transformants with correct insert produce gfp constitutively and the protein E is controlled by the heat sensor.
![](https://static.igem.org/mediawiki/2017/8/8b/T--ETH_Zurich--Cell_Lysis_phase_4a.png)
The double transformation (grown at 37 °C) yielded green colonies, which shows successfull inhibition of protein E at 37 °C (colonies are not lysed) and successful insertion of the protein E gene (+ const. promotor) between PLux and gfp (constitutive green at 37 °C)
protein E RBS library variant selection
All fluorescent colonies were picked and inoculated to a 96 well plate and grown overnight (16 h) to stationary phase at 37°C. Continuing with the 96 well format, the samples were inoculated into a fresh 96 well culture plate (OD 0.1) and grown to OD 0.4. At this point the cultures were split to fresh plates (flat transparent bottom) and induced at 37 °C and 45 °C for 3 h. The OD was measured from the beginning of the OD 0.1 culture to track the growth curve during induction. The 4 most promising variants were selected for the next experiment. They were restreaked to obtain multiple single clones for triplicate measurements.
Triplicate measurements of the best 4 variants
![](https://static.igem.org/mediawiki/2017/b/b6/T--ETH_Zurich--celllysisplots.png)
Experiment was performed according to the protocol with protein E and TlpA RBS library variants. The protein E RBS variants were sequenced and compared to the calculated translation initiation rates:
We could show that the heat sensor effectively induces protein E expression with 3 h of induction at 45 °C. The variant C has a very tight repression caused by the engineered TlpA RBS. This transformant unfortunately did not combine the RBS C (TlpA) with a strong protein E translation initiating RBS variant.
References
- Contois, D. E. "Kinetics of bacterial growth: relationship between population density and specific growth rate of continuous cultures." Microbiology 21.1 (1959): 40-50. doi: 10.1099/00221287-21-1-40