Part:BBa_K4016011

TCE

This part is an improvement of the exsisting TetOn-3G(Part:BBa_K1431301) designed by Rifei Chen, Yushan Zhang of iGEM14_SUSTC-Shenzhen .

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. TCE is a tetO7 promoter consists of seven direct 19-bp tet operator sequence (tetO) repeats upstream of a minimal CMV promoter (PminCMV). tetOs were separated by a 17bp flanking sequence, which was essential to the dox responsiveness of this promoter. The tetO constitutively binds tetR, it also binds to transcriptional activator tet-on 3G under the presence ofdoxycyline This promoter should be constructed just upstream of a Kozark consensus ribosome binding site to enhance the expression level of GOI.

Characterization

This part is validated through 4 ways: 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.

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]

Fuctional test: Improvement of an exsisting part

This part is an improvement of the exsisting TetOn-3G(Part:BBa_K1431301) . The improved TCE promoter shares a similar nucleic acid sequence to the original promoter in BBa_K1431301, with only a few nucleotides different on the flanking regions of tetO. However, these differences were sufficient to generate folds of changes on dox responsiveness.

The validation methods and results are as follows.

Method

We used SEAP assay to validate the improvement.SEAP reporter gene was constructed downstream of original TRE3G promoter and TCE promoter as BBa_K1431301-SEAP and BBa_K4016011-SEAP.. Therefore the SEAP activity in the culture medium could be used to report the activation of the promoters.

Meanwhile, we constructed tetR-3G into pcDNA3.1 vector to obtain pcDNA3.1-tetR-3G. TetR-3G can specifically bind to tetO and turn on the transcription of its downstream genes SEAP. SEAP activity could be measured witha para-Nitrophenylphosphate (pNPP) based assay, as SEAP catalyzes the reaction from pNPP to para-Nitrophenol, this new product absorbs light at 405 nm and can be measured via photometry.

Either BBa_K1431301-SEAP encoding plasmid or BBa_K4016011-SEAPencoding plasmid were co-transfected with tetR-3G encoding plasmid into HEK-293 cells. Cells were cultured for 6h before adding 500 ng/ml doxycycline, cells transfected with the same plasmids without doxycycline treatment were used as control. SEAP activity was detected 24/48 hours after transfection.

Here are the protocols:

• 1.Cell transfection and stimulation

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

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

HEK-293T cells co-transfected with tetR-3G expressing plasmid and either BBa_K4016011-SEAP or BBa_K1431301-SEAP plasmid showed significantly boosted SEAP production upon 24h or 48 h of doxycycline induction comparing to their respective control group, in which SEAP activities were below detection limit. This proves that both promoters did initiate the transcription of downstream gene. Intriguingly, under doxycycline induction, HEK-293T cells co-transfected with BBa_K4016011-SEAP and tetR-3G expressing plasmids showed ~30 times higher SEAP activity comparing to cells transfected with same dose of BBa_K1431301-SEAP and tetR-3G plasmids, showing a significantly improved performance of BBa_K4016011 comparing to Part:BBa_K1431301.

Figure1. Comparation of doxycycline responsiveness between the improved version and the original version of tetO7 promoters. (A)SEAP activity in the culture medium collect from cells 24 hours after 500 ng/ml doxycycline induction. (B)SEAP activity in the culture medium collect from cells 48 hours after 500 ng/ml doxycycline induction. BDL stands for below detection limit. Data represent mean ± s.e.m. (n=3 biological replicates)

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).