Difference between revisions of "Part:BBa K2969021"

Revision as of 21:33, 28 September 2024

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

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.

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.</p>

Sequence and Features