BBa_K567010

BBa_K567010 Version 1

Component

Source:
http://parts.igem.org/Part:BBa_K567010
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Yunfeng Ruan and Chunying Li
Date created: 2011-09-28 11:00:00
Date modified: 2015-05-08 01:12:43

PT7-Luc-8AGG



Types
DnaRegion

Roles
engineered_region

Reporter

Sequences BBa_K567010_sequence (Version 1)

Description

This biobrick is constructed by putting modified enzyme luciferase under promoter T7 and controlled by lac operator. 8 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNAArg (BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002).
Cell is cultured in 50ug/ml kanamycin 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA.
Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes.
Amount of bioluminescence produced can be detected using luminometer.

Notes

PCR is used to insert the 8 AGG codons into the gene after ATG.

Source

T7 promoter is derived from pET28a. Wild type Luciferase from BBa_I712019.

Sequence Annotation Location Component / Role(s)
ATG
8AGG
luciferase-8AGG
PT7
lac operator
rbs
89,91
95,118
89,1774
2,18
21,44
75,81
start_codon feature/start
feature/mutation sequence_alteration
CDS feature/cds
promoter feature/promoter
operator feature/operator
feature/rbs ribosome_entry_site
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synbiohub#topLevel
BBa_K567010/1