Difference between revisions of "Team:USP UNIFESP-Brazil/Proof"
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<p class="fig-label">Image 2 - Codon optimized mCherry spectrum of excitation and emission</p> | <p class="fig-label">Image 2 - Codon optimized mCherry spectrum of excitation and emission</p> | ||
<p class=black>In order to visually know if our cassette was expressing mCherry, we used a fluorescence microscope. Chloroplasts usually form a “U” shape around the cell with the nucleus in the middle. Apparently and for some reason, mCherry has accumulated near the nucleus as can be seen in the images below: </p> | <p class=black>In order to visually know if our cassette was expressing mCherry, we used a fluorescence microscope. Chloroplasts usually form a “U” shape around the cell with the nucleus in the middle. Apparently and for some reason, mCherry has accumulated near the nucleus as can be seen in the images below: </p> | ||
| − | <img src= | + | <img src="https://static.igem.org/mediawiki/2016/c/c1/T--USP_UNIFESP-Brazil--mCherry_microscopiacontrole.png" width= 450px> |
| − | <img src= | + | <img src="https://static.igem.org/mediawiki/2016/8/80/T--USP_UNIFESP-Brazil--mCherry_microscopiamcherry.png" width=450px> |
<p class=black>Figure 5: Fluorescence microscopy of C. reinhardtii. A - Measuring mCherry fluorescence. B- Measuring chlorophyll fluorescence. C - Open field image. D - Superposition of A,B and C. </p><br> | <p class=black>Figure 5: Fluorescence microscopy of C. reinhardtii. A - Measuring mCherry fluorescence. B- Measuring chlorophyll fluorescence. C - Open field image. D - Superposition of A,B and C. </p><br> | ||
<img src=https://static.igem.org/mediawiki/2016/b/b0/T--USP_UNIFESP-Brazil--mCherryfluor.gif width=500px><br> | <img src=https://static.igem.org/mediawiki/2016/b/b0/T--USP_UNIFESP-Brazil--mCherryfluor.gif width=500px><br> | ||
Revision as of 00:03, 20 October 2016
AlgAranha Team USP_UNIFESP-Brazil
The core iGEM idea was not only to produce spider silk proteins and antibiotics. But doing all this in microalgae!Thus, we also aimed to expand the tools and improve Chlamydomonas reinhardtii as a chassis for synthetic biology, characterizing a system for expression and secretion of recombinant proteins! We were able to genetically transform through electroporation and successfully express a reporter protein, mCherry, in Chlamydomonas and detect its fluorescence. Our composite part, BBa_K2136010 will be of great value for any other teams working with microalgae in iGEM’s future!
Even though this organism offers exciting possibilities, as well as many other photosynthetic chassis, a limited number of parts is available for synthetic biology work in the registry. Our motivation was to change this lack of parts and contribute to their improvement for the synbio community!
One of our goals within iGEM was to develop a “Chlamydomonas molecular toolkit”, with basic parts for everyone’s use. We had a construct, BBa_K2136010, synthesized by IDT gBlocks technology which included:
- An enhancer/promoter construct for transcription, BBa_K2136013
- A resistance gene, selection marker, against the antibiotic zeocin, BBa_K2136014
- A self cleaving peptide from foot-and-mouth disease virus, BBa_K2136017
- A signal peptide which enable secretion of the protein of interest, BBa_K2136018
This construct was already assembled through an scarless method with mCherry codon optimized coding sequence BBa_K2136016 and a Chlamydomonas terminator, BBa_K2136015
.The clones were then selected in Petri dishes containing zeocyn and screened in 96 well plates for mCherry fluorescence (excitation at 575nm, emission at 608nm) in a setup like this:
Image 0 - Setup of transformant Chlamydomonas screening
Even though the vast majority of the cells were productive, there was a variation in great part, due to the random assembly in C. reinhardtii’s genome. This, hopefully, will be surpassed with the development of new expression methods in the future (perhaps even by iGEM teams!). Here we show the expression profile during growth of the best 5 clones in our first and second screening:
Image 1 - mCherry emission/excitation in different cells in our first screen
Image 2 - mCherry emission/excitation in different cells in our second screen
The excitation/emission fluorescence spectrum of the mCherry supernatant (without cell lysis, indicating secretion) was also characterized, not only confirming the detection, but improving characterization of this protein (BBa_J06505 and BBa_K2136016 )in the context of iGEM.
Image 2 - Codon optimized mCherry spectrum of excitation and emission
In order to visually know if our cassette was expressing mCherry, we used a fluorescence microscope. Chloroplasts usually form a “U” shape around the cell with the nucleus in the middle. Apparently and for some reason, mCherry has accumulated near the nucleus as can be seen in the images below:
Figure 5: Fluorescence microscopy of C. reinhardtii. A - Measuring mCherry fluorescence. B- Measuring chlorophyll fluorescence. C - Open field image. D - Superposition of A,B and C.
Figure 6: 3D Fluorescence microscopy of C. reinhardtii. Chlorophyll is fluorescing in green and mCherry is fluorescing in red.
Finally, we checked the hability of our system to secrete proteins through the signal peptide (BBa_K2136018) through a home made filter, accordingly to our Human Practicesprinciples. We developed a system using a 532nm green laser pointer and a red filter to probe directly our centrifuged cells.
Image 2 - Laser and filter setup
In order to check the reliability of our systems, we compared the emission spectra for the 532nm green laser, the absorption spectra of the Rosco E-Colour #125: Deep golden Amber filter and the excitation/emission spectra for the mCherry protein:
Image 2 - Laser, filter and mCherry spectra superposition
Thus, the expected results were the following:
Image 2 - Theoretical results of "homemade" fluorimeter
And we achieved it in practice! Corroborating, one more time, our proof of concept, implementing an efficient protein expression and secretion system for Chlamydomonas for the first time in iGEM!!
Image 2 - Practical results of "homemade" fluorimeter
