BBa_K731700

BBa_K731700 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K731700
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
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BBa_K731700/1