Composite

Part:BBa_K1789012

Designed by: Jiaqi Sun   Group: iGEM15_NUDT_CHINA   (2015-09-14)

P+R

This part is a promoter. It has a strong RBS restriction site following it which can guarantee the high efficiency of our project.

Usage and Biology

This part is a promoter which is associated with a RBS(Ribosome binding site). It’s used to be a part which both has the function of the promoter and Ribosome binding site. The Plac promoter starts the whole work and the rbs30 with it ensure the high efficiency of ribosome binding.This part can improve the efficiency of our construction work we can use this part instead of linking parts every time we construct a plasmid.


1.RBS has been used to quantify translational coupling in E. coli synthetic operons.(2013, 2(6), ACS Synthetic Biology). 2.Some people are researching the effect of mutations in the lac promoter and catabolite repression of the lac operon. (Yudkin, M. D., 2005)

Sequence and Features

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Experimental Validation

Sequencing

This part is sequenced as correct after construction.

Enzyme digestion test

A double-enzyme digestion test was implemented to verify this part.

P+r酶切.jpg

The length of the digested DNA fits the design.

Fluorescence Analysis

The function of this part was examined by the GFP assay. The Plac+RBS30+GFP+Ter plasmid transfected E.coli BL21 (DE3) was cultured and IPTG induced. The groups without IPTG supplement were set as negative controls. Subsequently, the fluorescence intensity (abbreviated as FI, Ex: 488 nm; Em: 538 nm) were determined by the Fluoroskan Ascent FL (Thermo Scientific) every 2 hours.

Function of Plac+RBS30.jpg

Figure 2. Evaluation of the Plac+RBS30 by split GFP assay. The green fluorescence (Ex: 488 nm; Em: 538 nm) of split GFP was detected every two hours after IPTG induction. Relative fluorescence intensity was calculated with normalization of OD600 value. This experiment was run in three parallel reactions, and the data represent results obtained from at least three independent experiments. **p<0.01, *0.01<p<0.05.

Functional Parameters

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Categories
Parameters
None