Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Giacomo Giacomelli, Anna Depetris
Date created: 2012-08-13 11:00:00
Date modified: 2015-05-08 01:13:06
Platform for terminators analysis under the control of T7 promoter
| Types | DnaRegion |
| Roles | plasmid_vector Plasmid_Backbone |
| Sequences | BBa_K731700_sequence (Version 1) |
Description
We used this part to test terminators: the two subsequent proteins allow to quantify the termination efficiency as the ratio between Venus's and Cherry's fluorescence.The vector is pET21b, that contains lacI, t7 promoter and ampicillin resistance gene.
The two fluorescent proteins have partially overlapping emission spectra, thus the fluorimetric measures should be taken with these parameters, to avoid interference between the two:
Venus excitation: 485nm, Venus emission: 528nm
Cherry excitation: 528nm, Cherry emission: 615nm
We inserted the prefix-suffix linker between the two proteins, to allow terminator characterization.
Notes
Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers.For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics.
Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers.
For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics.
Source
It's made from a Roberta Lentini's construct (from Mansy lab) that consist of pET21b with the two proteins and a 20bp linker between them.That construct was mutated two times to eliminate illegal restriction sites; prefix-suffix linker was added by insertion PCR.
| Sequence Annotation | Location | Component / Role(s) |
| A206K VENUS T7 TERMINATOR ampicillin resistance gene lacI T7 PROMOTER lacO mCHERRY | 36,752 843,890 1711,2370 4562,5653 6031,6050 6050,6072 6119,6829 | CDS feature/protein sequence_feature feature/misc CDS feature/protein feature/protein CDS feature/promoter promoter feature/operator operator feature/protein CDS |