constitutive promoter of E. coli from EcoFlex kit

来源: E. coli DH10β
功能: promoter

Summary

A range of constitutive promoter were created by randomizing the E. coli σ70 Anderson promoter collection

基本信息

定量方法1 Cell populations were diluted into 1X phosphate buffered saline (2:1000) and loadedfor detection into a S3e Cell sorter (Biorad, CA, USA). GFP detection (FL1, Excitation 488 nm, Emission 525/30 nm) for approximately 48 000 cells per sample, was carried out and analyzed using FlowJo (v10.1r5) software (FlowJo, LLC, Oregon, USA).
定量方法2 Single colonies were picked in triplicate and grown overnight at 30 °C in a 24-well plate in 2 mL of 2YT medium with antibiotics. Cell density was measured in a CLARIOStar plate reader at 600 nm and cells were subcultured into 100 μL of 2YT with antibiotics in a 96-well Greiner plate to a starting OD600 of 0.05. Plates were sealed with a BreatheEasy membrane (Sigma) and grown at 30 °C for 6−12 h at 600 rpm. OD600, GFP and mCherry measurements were recorded every 10 min (see SI text). Data analysis of characterization data was quantified as described previously with one modification.27 Rates of GFP and mCherry production were quantified during exponential phase from an average of triplicate data over a 1 h time period. For cell-free activity measurements only, mCherry fluorescence accumulates significantly later than GFP and was measured at a different time point.
培养条件 Routine bacterial growth was performed at 37 °C in LB broth, with agar (15 g/ L),carbenicillin (100 μg/ mL), kanamycin (50 μg/ mL) or chloramphenicol (35 μg/ mL) as needed.
报告基因 GFP and mCherry
诱导剂
提交人 Yan Xing;Zhao Wenfang

元件序列

元件名 序列 序列长度
Bba_J23114
TTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGC
35
SJM903
CTTATAGCTAGCTCAGTCCTTGGGATTATGCTAGC
35
SJM911
TTGACAGCTAGCTCAGTCCTTGGTACTGTGCTAGC
35
SJM902
TTTACAGCTAGCTCAGTCCTAGGGATTATGCTAGC
35
SJM915
TTTATGGCTAGCTCAGTCCTTGGTATTATGCTAGC
35
SJM908
TTTATAGCTAGCTCAGCCCTTGGTATTATGCTAGC
35
SJM901
TTTACAGCTAGCTCAGTCCTAGGTATAATGCTAGC
35
SJM910
TTGATGGCTAGCTCAGTCCTTGGTATTATGCTAGC
35
SJM912
TTGATAGCTAGCTCAGTCCTAGGTACTATGCTAGC
35
Bba_J23108
CTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC
35
Bba_J23100
TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC
35
SJM905
TTTATAGCTAGCTCAGTCCTTGGGATTATGCTAGC
35
SJM906
TTGATGGCTAGCTCAGTCCTAGGGATTGTGCTAGC
35

References

1. Simon John Moore; Hung-En Lai; Richard Kelwick; Soo Mei Chee; David Bell; Karen M Polizzi; Paul S Freemont;  (2016). "EcoFlex - A Multifunctional MoClo Kit for E. coli Synthetic Biology."  pubmed