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Cell Lysis Experiments

This is a detailed experiment page dedicated to an individual function. To access other experiments, go to our Experiments page. To get a quick glimpse at all of our achievements, check out Results.

Introduction

CATE needs to release the previously accumulated Anti-Cancer Toxin to the extracellular space. We implemented the cell lysis mechanism from bacteriophage Phi X174. It 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 with the heat sensor and subsequent GFP release to the supernatant.

For more details about the lysis mechanism, go to Cell Lysis.

Phase I: Initial System Design

The cell lysis mechanism of phage Phi X174 needs a single gene called 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.

Lysis Plasmid Illustration — Figure 2. Cell Lysis test plamids. AHL inducible protein E is placed on a pSEVA291 vector. PF and SF are abbreviations for BioBrick Prefix and BioBrick Suffix restriction sites. RS1-RS4 are restriction sites that we introduced for subsequent cloning.

Initially, no transformants could be obtained. This was probably due to a high leakiness of the P_TlpA, which lead to enough expression of protein E to lyse all successful transformants.

CONCLUSION: We knew now that the protein E must be regulated by a very tight promotor. We used this knowledge to engineer the Heat Sensor to a low base level expression of the regulated gene. Read here how we engineered leakiness of the P_TlpA.

Phase II - Optimization of co-transformation efficiency

Initially, it was not possible to transform a protein E, regulated by the Heat Sensor into E. coli. Therefore we reduced the translation initiation rate of the protein E RBS.

Protein E RBS library creation

We constructed a ribosome binding site library to find variants expressing little enough protein E to avoid lysis from leaky expression, but still enough to lyse the cells when the expression of protein E is induced. The RedLibs (https://www.nature.com/articles/ncomms11163) algorithm was used.

We used the RedLibs algorithm to design a RBS library that would allow us vary protein expression and screen for improved variants. Therefore, we initially calculated the TIR values for a fully degenerate RBS library with 8 times N (fully degenerate base) at the positions -13 to -5 upstream of the ATG start codon using the Salis RBS calculator [A. Espah Borujeni, A.S. Channarasappa, and H.M. Salis, "Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites", Nucleic Acid Research, 2013 and H.M. Salis, E.A. Mirsky, C.A. Voigt, Nat. Biotech., 2009]. This library with 65’536 variants would be too large to efficiently screen and contain too many unfunctional RBS sequences. Therefore, we used the RedLibs algorithm to reduce the library to smaller size and distribute it’s values uniformly[Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort https://www.nature.com/articles/ncomms11163]. The algorithm then provided us with a partially degenerate sequence, that could be implemented by a single cloning step and codes for an as uniform as possible distribution of TIR values.

Distribution of TIR values calculated with RedLibs. Most sequences have a low TIR and should therefore enable transformants to grow despite some leakiness of the promotor.

The degenerate primers were ordered 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.

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. 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