Difference between revisions of "Part:BBa K2328027"

Latest revision as of 04:40, 10 October 2019

smURFP I + Linker A + RBS I + HO-1 I

Sequence and Features

Improvements

Group: TJUSLS_China 2019
Author: Ruoming Sun
Summary: We mutated the tyrosine of smURFP at position 56 to arginine.This change led to a significant increase in smURFP's ability to bind to the small molecule BV.
We co-expressed smURFP and Biliverdin-producing protein heme oxidase (Heme Oxygenase-1, HO-1) in E. coli and then purified the smURFP protein that binds BV small molecules. We used wild-type smURFP as a control and measured the absorbance at specific wavelengths of proteins, small molecule BV, and smURFP-BV complexes by ultraviolet spectrophotometer (280 nm, 388 nm, 642 nm, respectively). We normalized the wild type and mutant absorbance at 280 nm. Through data analysis, we found that the ratio of small molecule BV binding to protein in the mutant complex was significantly greater than that of wild type, which significantly demonstrated that the ability of the mutant to bind small molecules BV had been greatly improved.
New parts: BBa_K2933285.

Figure 5. smURFP, small molecules BV and BV- smURFP complexes were quantified by measuring the absorption values of wavelengths of 280 nm, 388 nm and 642 nm, respectively. (a)Absorption values of the wild type smURFP-BV complexes solution. (b)Comparison of absorption values of the wild type smURFP-BV complexes and the mutation smURFP(Y56R)-BV complexes.

Usage

It’s a co-expression system. smURFP (small ultra-red FP) is an important part in our group. It is desirable for our in vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP. HO-1 is the gene of the precursor of biliverdin. HO-1 can use the materials of the E.coil to produce biliverdin. So we want to construct a plasmid which can both express the smURFP gene and HO1 gene. Through this contruction, we can achieve the co-expression in the E.coil. Both the smURFP and biliverdin are produced by E.coil, so they can connect directly within the E.coil to produce fluorescence under the wavelength of 642nm without to add BV additionally. Besides, smURFP I and HO-1 I are both the codon-optimized version for Escherichia coli.

Biology

In order to produce fluoresce, smURFP must be combined with biliverdin (BV) .So one of our method is co-expression. Because the HO-1 needs to use oxygen to produce BV, it is adoptable in E.coil which is a kind of facultative anaerobic bacteria. And the HO-1 gene is from the Block Library. Through this contruction, we can achieve the co-expression in the E.coil. Both the smURFP and biliverdin are produced by E.coil, so they can connect directly within the E.coil to produce fluorescence under the wavelength of 642 nm.

Reference

[1] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.

[2] Dong Chen, Jason D Brown, Yukie Kawasaki, Jerry Bommer and Jon Y Takemoto. Scalable production of biliverdin IXα by Escherichia coli. [J].BMC Biotechnology, 2012.

Results

Co-expression in E.coli BL21

Fluorescence detection with Microplate Reader

Plasmid pET28b with smURFP and HO-1 gene were transformed into E. coli BL-21. We used this induced bacteria to confirm the fluorescence and data showed a relatively high value, as shown in table 1.

Table 1. Result of Microplate Reader in the black 96-well plate. Tube 1 and 2 are experimental group, and tube 3 is the control group.

Fluorescence imaging with FCFM

Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.

Figure 2. The result after induction, the upper one is the control group, and the inferior one is the experimental group.