BBa_K1833999

BBa_K1833999 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K1833999
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Konstantin Borisov
Date created: 2015-09-14 11:00:00
Date modified: 2015-09-16 07:58:33

T7 promoter as annealed oligos



Types
DnaRegion

Roles
Regulatory

promoter

Sequences BBa_K1833999_sequence (Version 1)

Description

This is a part used as a building block for T7 driven expression. However, this part uses a special method with the following synthesized nucleotides:

pT7sense: aattcgcggccgcttctagataatacgactcactatagggagaa
pT7antisense: ctagttctccctatagtgagtcgtattatctagaagcggccgcg

The following method can be used to insert a T7 promoter before a desired Biobrick part:

1. Cut the desired plasmid (which should contain an RBS and cds at minimum) with EcoRI and XbaI. Purify the plasmid with a method of your choice (both gel purification and PCR purification kits from Qiagen serve to remove the short undesired oligonucleotide from the digestion).
2. Be sure that your oligos have 5' phosphorylation (treat unphosphorylated oligos with T4 Polynucleotide Kinase). Then, anneal the two oligos by slowly cooling from 98C to room temperature.
3. Finally, ligate the cut plasmid with the annealed oligos by using a 1:10 molar ratio of plasmid to insert.
4. Transform the ligation reaction into E. coli, and plate.
5. Pick colonies, and verify the success of the cloning with sequencing or PCR. (Doing a diagnostic digest is not recommended as the size of the original plasmid and the desired plasmid are very similar.)
6. Add a composite part to the iGEM registry by using this part with the original protein coding part.

Notes

When ligating with a cut plasmid, this leaves a Biobrick scar between the T7 promoter and original Biobrick part.

Source

Synthesized oligonucleotides as described on main page.

Sequence Annotation Location Component / Role(s)
pT7
Oligo scar
1,23
24,29
feature/promoter promoter
feature/scar
igem#experience
None
 
igem#status
Planning
 
synbiohub#ownedBy
user/james
 
synbiohub#ownedBy
user/myers
 
synbiohub#topLevel
BBa_K1833999/1