Difference between revisions of "Part:BBa K2136016"
| Line 4: | Line 4: | ||
Codon-optimized for Chlamydomonas reinhardtii mCherry | Codon-optimized for Chlamydomonas reinhardtii mCherry | ||
| + | |||
| + | [[Part:BBa_K2136010]] | ||
| + | |||
| + | mCherry is a red fluorescent protein used as a reporter. It is based on a fluorescent protein that was originally isolated from ‘’Discosoma sp’’. and it’s being largely used due to its colour and photostability compared to other monomeric fluorophores. Another important property is that, with a system such as the one in [Part:BBa_K2136010]] secretion cells partially secrete mCherry. Therefore, it’s possible to monitor, in real-time, the kinetics of the process evaluated with aliquots of the cultivation medium or the biological material in study [1]. | ||
| + | The codon optimized mCherry for ‘’Chlamydomonas reinhardtii’’comes from the biobrick BBa_J06504 and it was improved to work specially with C. reinhardtii, a microscopic algae used as model organism to study photosynthesis, cellular division, flagellar biogenesis, and, more recently, mitochondrial function [2]. | ||
| + | Our team used this codon optimized mCherry to test the promoter activity and the expression capacity of the our new plasmid for microalgae transformation gBlock1 ([Part:BBa_K2136010]] ) . | ||
| + | |||
| + | Methods | ||
| + | Because not all tRNA are expressed equally, specially across species, a particular DNA sequence can be codon optimised to match the most prevalent tRNAs of the host cell, improving the efficiency of protein translation[REF]. So here are the changes that we made in the DNA sequence using the software GeneArt from Life Technologies: | ||
| + | |||
| + | For a better characterization of mCherry we’ve measured its excitation and emission spectra using a Tecan M200 Pro Microplate reader. In the 96 well plate we’ve measured the excitation and emission spectra of transformed C. reinhardtii supernatant, wild C. reinhardtii supernatant, water, TAP medium, transformed C. reinhardtii with spent TAP, wild C. reinhardtii with spent TAP, washed transformed C. reinhardtii with fresh TAP and washed wild C. reinhardtii with fresh TAP. | ||
| + | Those wells were monitored using a Tecan M200 Pro Microplate reader. For mCherry fluorescence detection we used excitation wavelength at 575 nm and emission at 608 nm, for inactive mCherry we used excitation wavelength at 410 nm and emission at 461, for Chlorophyll fluorescence we used 440 nm for excitation and 680 nm for emission. We also measured the absorbance at 750 nm for cellular concentration. | ||
| + | |||
| + | <html> | ||
| + | <img src="https://static.igem.org/mediawiki/2016/0/09/T--USP_UNIFESP-Brazil--mCherry_spectra1.jpeg" width="900px" style="margin-bottom:20px; margin-top:0px;" /> | ||
| + | <center><p class="fig-label">Codon optimized mCherry spectrum of excitation and emission</p></center> | ||
| + | </html> | ||
| + | |||
| + | |||
| + | https://static.igem.org/mediawiki/2016/6/69/T--USP_UNIFESP-Brazil--mCherry_gel.jpeg | ||
| + | Image 1: Gel electrophoresis of mCherry | ||
Revision as of 00:01, 19 October 2016
Codon optimized mCherry for Chlamydomonas reinhardtii
Codon-optimized for Chlamydomonas reinhardtii mCherry
mCherry is a red fluorescent protein used as a reporter. It is based on a fluorescent protein that was originally isolated from ‘’Discosoma sp’’. and it’s being largely used due to its colour and photostability compared to other monomeric fluorophores. Another important property is that, with a system such as the one in [Part:BBa_K2136010]] secretion cells partially secrete mCherry. Therefore, it’s possible to monitor, in real-time, the kinetics of the process evaluated with aliquots of the cultivation medium or the biological material in study [1]. The codon optimized mCherry for ‘’Chlamydomonas reinhardtii’’comes from the biobrick BBa_J06504 and it was improved to work specially with C. reinhardtii, a microscopic algae used as model organism to study photosynthesis, cellular division, flagellar biogenesis, and, more recently, mitochondrial function [2]. Our team used this codon optimized mCherry to test the promoter activity and the expression capacity of the our new plasmid for microalgae transformation gBlock1 ([Part:BBa_K2136010]] ) .
Methods Because not all tRNA are expressed equally, specially across species, a particular DNA sequence can be codon optimised to match the most prevalent tRNAs of the host cell, improving the efficiency of protein translation[REF]. So here are the changes that we made in the DNA sequence using the software GeneArt from Life Technologies:
For a better characterization of mCherry we’ve measured its excitation and emission spectra using a Tecan M200 Pro Microplate reader. In the 96 well plate we’ve measured the excitation and emission spectra of transformed C. reinhardtii supernatant, wild C. reinhardtii supernatant, water, TAP medium, transformed C. reinhardtii with spent TAP, wild C. reinhardtii with spent TAP, washed transformed C. reinhardtii with fresh TAP and washed wild C. reinhardtii with fresh TAP. Those wells were monitored using a Tecan M200 Pro Microplate reader. For mCherry fluorescence detection we used excitation wavelength at 575 nm and emission at 608 nm, for inactive mCherry we used excitation wavelength at 410 nm and emission at 461, for Chlorophyll fluorescence we used 440 nm for excitation and 680 nm for emission. We also measured the absorbance at 750 nm for cellular concentration.
Codon optimized mCherry spectrum of excitation and emission
Image 1: Gel electrophoresis of mCherry