Part:BBa_K2969021

TCI

TCI is a temperature-sensitive variant of the bacteriophage λ repressor cI. It consists of N-terminal, C-terminal and the fragment between N-terminal and C-terminal whose function is the linkage. It is a cold-inducible transcription factor. When the temperature is below 35℃, the activity of TCI will gradually rising. When the temperature is above 35℃, the activity of TCI will remain at a low level.

Characterization

In 2019, UCAS-China developed a collection of thermosensitive parts with high-performance, versatility and robustness. Based on TCI transcription factor family and TlpA family, we collected five TCI and TlpA mutants and used sfGFP as reporter to build some heat-inducible ON-switches, which can open gene expression under high temperature. To characterize them quantitatively, we first characterized the performance of them by flow cytometer in Top10 strain.

As shown in Figure 1, most of the transcription repressors show sharp thermal transitions, especially TCI and TCI42, with more than 100-fold induction within 10 degrees Celsius. Their impressive performances make them candidate parts for our further circuit design.

Figure 1:The induction curves of the heat-inducible switches (TOP10)

What’s more, we also tested these heat-inducible ON-switch in the chassis E.coli Nissle 1917, a probiotic with more than 100 years of medical application, their robustness give us more confidence in the stability and preciseness of our ark. The result is shown in Figure 2.

Figure 2:The induction curves of the heat-inducible switches (Nissle 1917)

Reference

Piraner, D. I., Abedi, M. H., Moser, B. A., Lee-Gosselin, A., and Shapiro, M. G., Tunable thermal bioswitch for in vivo control of microbial therapeutics. DOI: 10.1038/NCHEMBIO.2233

TcI double-plasmids reporter system by Cambridge 2024

Designed by: Dhriti Krishna Rao, Cenyujia Wang, Jamie Terry
Uploaded by: Linhan Lei

Introduction

TcI is a thermolabile mutant form of the cI protein of bacteriophage lambda, which unbinds its operon at temperatures above 37 Celsius (Valdez-Cruz et al, 2010). We are aiming to design a system with two plasmids: one expressing TcI under the standard promoter, and the other expressing a fluorescent protein under the control of or TcI. The ideal system will express the fluorescent protein at temperatures above 37 Celsius. Successful construction of the double-plasmid will allow us to replace the fluorescent protein with any cds, and the expression of the selected cds will be strictly heat-sensitive.

Design

The double plasmid system uses parts mostly from the distribution kit. TcI is under the control of a constitutive Anderson promoter (BBa_J23119), and a double terminator B0015 is used in the system. We chose the BBa_J23119 promoter because the characterisation on the registry suggested a higher rate of transcription compared to the other tested promoters. We also used mRFP1 instead of eYFP because we wanted to visualise the fluorescence more easily against the yellow autofluorescence of the LB agar.

Figure 1 | Construction of 1p and 2p double-plasmids system.

Construction

The promoter and terminator were extracted from the kit by PCR, rather than transformation and miniprep. The plasmids were assembled by golden gate, and two types of transformations were performed. One involved each golden gate reaction being chemically transformed into DH5alpha, as is standard. In the interest of time, we also electroporated both reactions into BL21 at once and plated onto a double antibiotic plate. Both sets of transformations were successful as colonies grew on their respective single or double antibiotic plates. 1p and 2p V1 cells were heat shocked for 1 min, and V2 heat shocked for 30s. BL21 cells were plated onto plates: α β with no difference between the cells on each plate. As the BL21 double plasmid cells grew, we proceeded with thermal testing. However, following a colony PCR, we found that the 1p Golden Gate was unsuccessful and some parts were missing.

Figure 2 | Colony PCR of 1p from BL21 cells. The bands appear around 700 bp, and the expected band in all 4 samples is 953bp. Left bottom row: Colony PCR of 1p from DH5α cells. The bands appear around 700 bp, and the expected band in all 4 samples is 953 bp.

Figure 3 | Colony PCR of 1p V2 from DH5α cells. The bands appear around 1000 bp, and the expected band in all 6 samples is 1041 bp.

Testing

We tested the BL21 on the hot plate for 5 hours. At the beginning, the cells were white and remained white after 5 hours. Our positive control was the β2 pink colony as it was pink even at room temperature. Next, we put the plate in a 43℃ incubator for 1 hour. After growing overnight at room temperature, the colonies remained white. After growth at room temperature for another day, the colonies had a slight pink colour in the centre.

Figure 4 | Hot plate testing, cells remained white after 5h.

Figure 5 | Cells before and after 5h of hot plate testing and 1h in the 42℃ incubator. After growth overnight in the 37℃ incubator, the cells had a slight pink colour in the centre.

Following this test, we wanted to see if expression of mRFP1 could be increased by increasing the incubation time. After reading RDFZ-China 2018 iGEM team’s wiki (https://2018.igem.org/Team:RDFZ-China), we decided to incubate different colonies off the same plate for 24h.
6 colonies from each plate (α and β) were inoculated in liquid as well as plated on solid double antibiotic plates over the weekend at room temperature. After 65h, the colour of the cells were mostly white with some samples either with a pink centre or were fully pink(see table 1).

Table 1 | Colour of liquid and solid versions of each.

Plates were made with the same samples above and incubated at 24h at 4 different temperatures: 23℃ (room temperature in our lab), 30℃, 37℃ and 42℃.
After 24h, the 37℃ and 42℃ plates had grown a lot of colonies. A control plate as also incubated at room temperature, however after 24h, there were no colonies grown. Therefore, we chose to compare the plates against the room temperature 89h growth as the colony density was more compatible. Compared to the room temperature α plate that grew over the weekend, where only α2 was fully pink, all of the colonies on the α plate that grew in 37℃ for 24h were more pink. These plates suggested that temperature did have an effect on the expression of mRFP1.

Figure 6 | Left: Plates with TcI BL21 cells. The 30℃ did not seem to have many colonies after 24h, and the 23℃ had no colonies for all 12 samples. Right: All samples on the α plate that grew in 37℃ for 24 h were pink.

From the plates, it could be suggested that 37 and 42℃ had the greatest activation of expression of mRFP1. To try and distinguish between 37 and 42, we put them in the specific incubators for another 24h.

Figure 7 | Plates after 48 hrs.

After 48h, all 6 samples on plate α seem to have the highest fluorescence on either 37℃ or 42℃ plates. For some samples e.g. α3 and 9, more cells seemed to be fluorescent on the 37℃ plate. This aligns with AIS-China 2023 iGEM team’s characterisation of BBa_C0051, where fluorescence/OD600 peaks at 37℃. On the other hand, β10 seems to have the higher fluorescence at 23℃, and fluorescence decreases as temperature increases.

We wanted to test if the increase in fluorescence was due to temperature reducing TcI inhibition of mRFP1 expression or due to the faster cell growth. To do this, the liquid samples from over the weekend were diluted and reinoculated and transferred to the same 4 different temperatures. After 24h, an end point reading of fluorescence and OD600 was taken using a plate reader. The data was blank corrected and relative fluorescence was calculated by: fluorescence[sample]/(fluorescence[blank]*OD600)

Figure 8 | Plate reader results.

However, there were a few negative values in the OD600 reading. A suggestion for this outcome is the droplets on the film cover from overfilled wells.

References

1. Valdez-Cruz, Norma A., et al. "Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters." Microbial cell factories 9 (2010): 1-16.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]