Difference between revisions of "Part:BBa K1033901"
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[[Image:meffBlue1.png|500px|center]] | [[Image:meffBlue1.png|500px|center]] | ||
Fig.1 The fermentation broth of meffBlue Fig.2 Proteins after secondary ultrafiltration | Fig.1 The fermentation broth of meffBlue Fig.2 Proteins after secondary ultrafiltration | ||
| + | Conclusion: The cell pellet was collected by harvesting 50mL culture after 24h of induction followed by centrifugation at 4 degrees and 6000 rpm for 10min. Then, we performed ultrasonic disruption and collected the supernatant after centrifugation. The protein was purified and collected through ultrafiltration and affinity chromatography. | ||
Revision as of 17:12, 11 October 2022
meffBlue, blue chromoprotein (incl RBS)
This chromoprotein from the coral Montipora efflorescens, meffBlue (also known as Rtms5), naturally exhibits strong color when expressed. The protein has an absorption maximum at 592 nm giving it a blue color visible to the naked eye. Compared to many other chromoproteins, such as amilCP (BBa_K592009), amilGFP (BBa_K592010), spisPink (BBa_K1033932), asPink (BBa_K1033933) and aeBlue (BBa_K864401), the color development is slower. The color is readily observed in both LB or on agar plates after 24-64 hours of incubation. The protein meffBlue has significant sequence homologies with proteins in the GFP family.
Usage and Biology
This part is useful as a reporter.
Source
Montipora efflorescens. The protein was first extracted and characterized by Beddoe et. al. under the name Rtms5 (UniProtKB/Swiss-Prot: P83690.2). This version is codon optimized for E coli by Genscript.
References
[http://scripts.iucr.org/cgi-bin/paper?cy0089] Beddoe, Travis, et al. "The production, purification and crystallization of a pocilloporin pigment from a reef-forming coral." Acta Crystallographica Section D: Biological Crystallography 59.3 (2003): 597-599.
[http://www.sciencedirect.com/science/article/pii/S0969212603000285] Prescott, Mark, et al. "The 2.2 Å crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation." Structure 11.3 (2003): 275-284.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
Team:NJTech_China 2022
Hoping to make a useful contribution for future iGEM teams,we completed the experimental characterization of existing parts tsPurple, purple chromoprotein(incl RBS) and meffBlue, blue chromoprotein and provided new documents for both of them.
The meffBlue sequence (Part:BBa_K1033901) optimized for E. coli was incorporated into plasmid pET-29a(+), transformed into E. coli BL21 for characterization and measurement. We provided meffBlue with results and data based on protein expression and purification, TOF-Mass spectrometry, and Swiss-model.
Methods: SDS-PAGE, ultramicro spectrophotometer ,TOF-Mass Spectrometry, and Swiss-Model.
Results:
Fig.1 The fermentation broth of meffBlue Fig.2 Proteins after secondary ultrafiltration
Conclusion: The cell pellet was collected by harvesting 50mL culture after 24h of induction followed by centrifugation at 4 degrees and 6000 rpm for 10min. Then, we performed ultrasonic disruption and collected the supernatant after centrifugation. The protein was purified and collected through ultrafiltration and affinity chromatography.