Difference between revisions of "Part:BBa K592100"

Revision as of 08:01, 20 October 2019

Blue Fluorescent Protein (mTagBFP)

This part codes for the bright blue fluorescent protein mTagBFP. mTagBFP is a monomeric protein with a narrow fluorescence emission spectrum with a maximum at 456 nm. It has a tyrosine-based chromophore, giving it substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescent proteins with a histidine in the chromophore. Subach et. al. started with a red fluorescent protein (TagRFP) and converted it to a bright blue monomeric protein. See the article listed in source. The sequence has been codon optimized for expression in E coli by DNA 2.0.

Subach et. al. reported the following data:

Excitation peak: 399 nm. Emission peak: 456 nm. Maturation Half-Time (min): 13. Relative brightness to EBFP2: 1.82. Extinction Coefficient (M-1 cm-1): 52000. Quantum Yield: 0.63.

Usage and Biology

This part is useful as a reporter.

iGEM11_Uppsala-Sweden. Fluorescence on UV table. The images above show E coli constitutively expressing mTagBFP BBa_K592100 (blue) and YFP BBa_K592101 (yellow) illuminated on a UV table.

References

[http://www.ncbi.nlm.nih.gov/pubmed/18940671] Subach, O. M., I. S. Gundorov, et al. (2008). "Conversion of red fluorescent protein into a bright blue probe." Chem Biol 15(10): 1116-24.

Jiangsu_High_School 2019’s Characterisation

BBa_K592100 Blue Fluorescent Protein (mTagBFP)

The E. coli expressing blue fluorescent protein(mTagBFP) were cultured at 28 °C and the overnight bacterial solution was collected for subsequent measurement.

After ultrasonically disruption of the above bacterial solution ,5 ul of each was added to 4 kinds of 45 ul solution having a pH of 3, 5, 7, and 9, and the fluorescence was measured using varioskan flash of Thermo Scientific. The excitation wavelength/emission wavelength corresponding to each fluorescent protein is: BFP:402/457.

Figure Fluorescence of BFP under different pH. There were two kinds of BFP expressing in E.coli(BFP) and expressing in E.coli after ultrasonically disruption(BFP-UC). Under the same pH, the fluorescence of BFP was higher than BFP-UC. With increasing of pH( among pH3, pH5, pH7,), the fluorescence of BFP and BFP-UC all raised. When the pH increased from pH7 to pH9, the fluorescence of BFP and BFP-UC were all down.

The results showed that the fluorescent value of each fluorescent protein did not change significantly in acid and alkali circumstance before sonication, but after sonication, each fluorescent protein showed the highest fluorescent value at pH 7, and the fluorescent value decreased as the environment became more acidic.

Shanghai_HS_United 2019's characterization

BBa_K592100 Blue Fluorescent Protein (mTagBFP)

When the fluorescent protein is excited, an emission spectrum is generated, and the emitted light has different fluorescence intensities at different wavelengths. To explore this, we designed a full-wavelength scan of the blue fluorescent protein.

1. We transformed the plasmid contanining BBa_K592100-mTagBFP into bacterial competent cells separately, plated and cultured overnight at 37 °C.

2. On the evening of the next day, we picked single clone into a tube containing 5 ml LB which is called 0h. At the same time, we also inoculated competent DH5α strain into a tube containing 5 ml LB as a blank control.We culture bacterial cells at 37 ° C, 220 rpm. The sampling time point is 24h.

3. We took 100ul of the bacteria culture and measured the total fluorescence with a Thermo fluorescence microplate reader. We took 200ul of the ten-fold diluted bacterial solution and measured the OD600 with BioTek optical microplate reader. Total fluorescence is divided by OD600 to obtain fluorescence value per OD600.

Figure1. TagBFP containing strain RFU/OD600 at different emission Wavelength.

The K592100-mTagBFP RFU/OD600 of the strain varies with the emission wavelength. The overall curve showed a normal distribution and the RFU/OD600 of the strain reachs its maximum at 456 nm. The absorbance value of the blank control DH5α is close to zero.

These results indicate that the absorbance value of the strain is produced by the BFP protein expressed in the cell instead of other factors in the cell, and the BFP protein has the optimal excitation light wavelength 402nm and emission light wavelength 456nm.

Sequence and Features