Revision as of 17:25, 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 P_TlpA 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 P_TlpA 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 P_Lux. 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 P_Lux, 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 P_Lux 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.

TlpA RBS library variant selection

Single colonies were picked and inoculated to a 96 well plate and grown to a stationary phase. Continuing with the 96 well format, the samples were inoculated into a fresh 96 well culture plate and grown to OD 0.4. At this point the cultures were split and induced at 37 °C and 45 °C for 3 h. Samples were diluted in PBS and the fluorescence measured in a plate reader. The eight variants with the highest fold-change were selected for further experiments.

The best eight TlpA RBS variants were tested for fluorescence induction according to the protocol.

Figure 6. TlpA RBS Library variants on/off ratio. Distribution of obtained fluorescence expression of the library variants and their fold change. The fold changes have a high variance from 20 to 7000. This data suggests that different variants of the original RBS were compared. The new variants from the library generation yield higher fold changes than the parent variant which had a RBS with rather low translation initiation rate. The high fold change of H1 is caused by the low leakiness, not by extraordinary high expression.

Figure 7. The 96 well plate with the four technical replicates of the induction of gfp with the thermosensitive TlpA repressor. On top: 4 wells per column induced for 3 h at 37 °C, bottom: 4 wells per column induced at 45 °C. The positive control did not grow.

Triplicate measurements of the best 3 variants

Experiment was performed according to the protocol with TlpA RBS library variants H1, A9, C12 and D9. They 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

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

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          <figcaption>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. .</figcaption>
      </figure>
-           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
+     <p>
+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 P<sub>Lux</sub> and gfp (constitutive green at 37 °C)
+ <figure class="fig-nonfloat" style="width:500px;">
+        <img src="https://static.igem.org/mediawiki/2017/5/51/T--ETH_Zurich--1-11and2-2doubletransformationplate.jpeg">
+        <figcaption>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.</figcaption>
+    </figure>
 </p>