Difference between revisions of "Part:BBa K4016011"

Revision as of 18:50, 21 October 2021

TCE

We’ve designed TCE this year, improving TetOn-3G designed by Rifei Chen, Yushan Zhang of iGEM14_SUSTC-Shenzhen (Part:BBa_K1431301).

Usage and Biology

TCE(Tetracycline-Controlled Transcriptional Activation, also known as TRE-Tight) is a system of inducible gene expression systems for mammalian cells. TRE-Tight contains an MCS immediately downstream of the Tet-responsive Ptight promoter. cDNAs or genes inserted into the MCS will be responsive to the tTA and rtTA regulatory proteins in the Tet-Off and Tet-On systems[1-2], respectively. Ptight contains a modified Tet response element (TREmod), which consists of seven direct repeats of a 36-bp sequence that contains the 19-bp tet operator sequence (tetO). The TREmod is just upstream of the minimal CMV promoter (PminCMV), which lacks the enhancer that is part of the complete CMV promoter. Consequently, Ptight is silent in the absence of binding of TetR or rTetR to the tetO sequences. Note that the cloned insert must have an initiating ATG codon. In our Project, addition of a Kozak consensus ribosome binding site has improved expression levels.

Sequence and Features

Special Design

We have made a series of modification and functional improvements based on TetOn-3G designed by Rifei Chen, Yushan Zhang of iGEM14_SUSTC-Shenzhen (Part：BBa_K1431301). In addition, we also added a Kozak consensus ribosome binding site to improve expression levels. What’s more, target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (PTRE3G) will express high levels of GOI, but only when cultured in the presence of Dox, which is a synthetic tetracycline derivative. In the presence of Dox, Tet-On 3G binds specifically to PTRE3G and activates transcription of the downstream GOI. PTRE3G lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction. However, in our project, tetR will combine with tetO itself -- no induction of small molecules is required.

Experimenal Validation

This part is validated through 4 experiments: PCR, enzyme digestion, sequencing and functional test.

PCR

The PCR is performed with 2 x Phanta Max Master Mix.

F-Prime: 5’-ATTTCTCTATCGATAGGTACCCCGCTATCTAGACGAGTTTAC-3’

R-Prime: 5’-AGCAGCATGGTGGGCGAATTCCCTTTCGAGGGTAGGAAGTGGTA-3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose gel.

Enzyme Digestion

After the assembly the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The cutting procedure was performed with Hind III EcoR I restriction endonuclease bought. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose gel.

Sequecing

The plasmid was sequenced correct.

Fuctional test

1.Cell transfection and stimulation

• Material

24-well cell culture plates, DMEM, polyethylenimine (PEI) solution (1 mg/mL), Vortex oscillator

• Steps

① Seed approximately 5 x 104 cells into 24-well cell culture plates.

② Culture for 16 h before transfection.

③ Total plasmid mixes of 500 ng per well are mixed thoroughly in serum-free DMEM before a polyethylenimine (PEI) solution (1 mg/mL) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight).

④ The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.

⑤ Cells are then changed into fresh medium and applied with stimulus for before sampling and analysis assay.

• Note

To validate and optimize the gene circuits to reach the intended functionality and performance with high throughput, transient transfections are preferred. Also, reporters that are easy to be detected (such as SEAP and Luciferase) are also preferred during this process to evaluate the dynamics of the circuits

2.SEAP assay in vitro

• Material

Chemiluminescence-based SEAP assay kit (e.g., Sigma Aldrich; cat. no. 1177984200) buffer (2x): 20 mM L-Homoarginine hydrochloride, 1 mM MgCl2, 21% (v/v) diethanolamine, pH 9.8. Substrate for SEAP: 120 mM para-nitrophenyl phosphate (pNPP) in 2SEAP buffer. Transparent 96-well plates. Colorimetric plate-reader.

• Steps

① Sample 200 μL culture medium from each well, heat inactivate at 65 C for 30 min.

② During the heat inactivation procedure, warm up 2 SEAP buffer (100 μL/well) at 37 ℃.

③ Add 1/5 buffer volume of pNPP (20 μL/well) substrate into the 2x buffer to prepare the “Detection Mixture.”

④ Add 80 μL heated medium into the 96-well plate, add 120 μL Detection Mixture.

⑤ Measure absorption at 405 nm, 37 s per read for 10 reads.

⑥ Calculate enzymatic activity.

• Note

SEAP levels in culture supernatants can be quantified with a pnitrophenylphosphate-based colorimetric assay

Result

After the SEAP assay in vitro, we found the enzymatic activity of SEAP as the figure shows below.

Figure 1The experiment verifying TCE promoter is much more powerful than TetOn_3G. As we can see in this figure, the transcriptional effect of TCE is far stronger than TetOn-3G for the enzymatic activity (from co-transformation which BBa_K4016011 participated in) is much higher than others in varied conditions. This can definitely reflect our significant improvement on TetOn-3G.

Reference

[1] T. Das, A., Tenenbaum, L. & Berkhout, B. Tet-On Systems For Doxycycline-inducible Gene Expression. CGT 16, 156–167 (2016).

[2] Das, A. T., Zhou, X., Metz, S. W., Vink, M. A. & Berkhout, B. Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells. Biotechnology Journal 11, 71–79 (2016).