Difference between revisions of "Team:ETH Zurich/Experiments/Cell Lysis"

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Revision as of 19:47, 30 October 2017

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
Figure 1. The genetic mechanism of our TlpA heat sensor. TlpA dimers bind in the PTlpA region and repress transcription of the downstream gene. A temperature of 45 °C shifts the equilibrium of dimerization towards monomers, which don't bind in the PTlpA region. Transcription of protein E can therefore happen at 45 °C. Protein E molecules interfere with cell wall synthesis and lead to cell lysis. Previously accumulated toxins get released.

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
Figure 2. Cell Lysis test plamids. AHL inducible protein E is placed on piG17-1-006 (pSEVA291). PF and SF are abbreviations for BioBrick Prefix and BioBrick Suffix restriction sites. RS1-RS4 are restriction sites that we introduced for later cloning. This plasmid could not be transformed into bacterial cells probably due to high leakiness of the promotor.

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.

Figure 5. Heat inducible cell lysis test device. The plasmids contained RBS libraries to increase the chance of finding transformants with the right amount of cytoplasmic protein E. The best variant of the TlpA RBS (variant C) was also used for this transformation in parallel. .

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)

Figure 6. Double transformation of the heat inducible cell lysis test device (protein E RBS library and TlpA RBS library). A couple of green fluorescent colonies were obtained. They were tested in later experiments for inducible cell lysis.

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

Figure 7. protein E RBS library variants fluorescence ratio (supernatant/total). Four library variants were selected and induced with a heat shock of 45 °C (lower row). The Variant C has the lowest leakiness of protein E at 37 °C as expected. It is the engineered TlpA RBS and leads to a tight repression of protein E. The negative control consists of a constitutively expressed protein E without protein E. The variance on the gfp release has influence from the TlpA RBS and the protein E RBS, which are both different for variants 2, 4 and 13. Variant C has a known TlpA RBS Sequence.

Experiment was performed according to the protocol with protein E and TlpA RBS library variants. They protein E RBS variants were sequenced and compared to the calculated translation initiation rates:

The thermoswitch was now tight enough to repress the toxic protein E to enable transformant colonies to grow. It will be transformed together with a protein E RBS library containing plasmid, with the aim to find protein E RBS library variants with enough reduced translation initiation rate to survive.

Figure 8. TlpA-regulated gfp expression. left: Fluorescence/OD of the variants C12 (C), A9 (A), H1 (H) and the parent variant. right: fold changes. Different variants of the RBS of TlpA were induced and not induced in triplicates (three different colonies picked). They have a reduced leakiness compared to the parent RBS which lead to a higher on/off ratio.

To read more about each of these experiments, click on the buttons below. For a detailed protocol describing each experiment, visit Protocols.

Phase III - A: Combination of heat sensor and cell lysis

References

  1. 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