Difference between revisions of "Part:BBa K2136016"
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== Methods ==
 
== Methods ==
  
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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.
+
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:
−
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.
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<img src="https://static.igem.org/mediawiki/parts/7/73/T--USP_UNIFESP-Brazil-result_Screen_1_mCherry.png" width="450px" style="margin-bottom:20px; margin-top:0px;" />
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<p class="fig-label">Image 1 - mCherry emission/excitation in different cells</p>
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<table class="tg">
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<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;" />
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  <tr>
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<center><p class="fig-label">Image 2 - Codon optimized mCherry spectrum of excitation and emission</p></center>
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    <th class="tg-031e">Before Optimization</th>
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<p><br></br></p>
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    <th class="tg-yw4l">After Optimization</th>
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  </tr>
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    <td class="tg-yw4l">ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATAA</td>
 +
    <td class="tg-yw4l">ATGGTGTCCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGAGCCCCCAGTTCATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACAGCAGCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCAGCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCAGCAGCGAGCGCATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGCGCCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTGAACATCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGCTGAGGGCCGCCACAGCACCGGCGGCATGGACGAGCTGTACAAGTAA</td>
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  </tr>
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</table>
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</html>
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We successfully amplified the codon optimized mCherry, which has 711 bp, as can be seen in image 1.
 
<html>
 
<html>
 
<img src="https://static.igem.org/mediawiki/2016/6/69/T--USP_UNIFESP-Brazil--mCherry_gel.jpeg" width="400px" style="margin-bottom:20px; margin-top:0px;" />
 
<img src="https://static.igem.org/mediawiki/2016/6/69/T--USP_UNIFESP-Brazil--mCherry_gel.jpeg" width="400px" style="margin-bottom:20px; margin-top:0px;" />
 
<p class="fig-label"> Image 3 - : Gel electrophoresis of mCherry+pSB1C3 construct</p>
 
<p class="fig-label"> Image 3 - : Gel electrophoresis of mCherry+pSB1C3 construct</p>
 
</html>
 
</html>
 +
Image 1: Gel electrophoresis of mCherry
 +
 +
After ligating the sequence in the pJP22, we wanted to find the best way to transform C. reinhardtii, we tested Sapphire Blue, TAP medium and water as buffers during the electroporation. Then, cells were plated in Agar-TAP medium Petri dishes with different concentrations of Zeocin (an antibiotic from Bleomycin family) because the plasmid used had a sequence for Bleomycin resistance.
 +
 +
<html>
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<img src="https://static.igem.org/mediawiki/2016/6/6c/T--USP_UNIFESP-Brazil--mCherry_placaA5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
 +
<img src="https://static.igem.org/mediawiki/2016/e/ef/T--USP_UNIFESP-Brazil--mCherry_placaA10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
 +
<br>&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Figure 2: pJP22 mCherry transformants - Zeocin 5 ug/mL &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Figure 3: pJP22 mCherry transformants - Zeocin 10 ug/mL<br>
 +
<img src="https://static.igem.org/mediawiki/2016/d/d9/T--USP_UNIFESP-Brazil--mCherry_placaM5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
 +
<img src="https://static.igem.org/mediawiki/2016/7/77/T--USP_UNIFESP-Brazil--mCherry_placaM10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" /> <br>
 +
&nbsp; &nbsp; &nbsp; &nbsp; Figure 4: pJP22 mCherry transformants - Z5 (Water Transformed)&nbsp; &nbsp;&nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Figure 5: pJP22 mCherry transformants - Z10 (Water Transformed)
 +
<img src="https://static.igem.org/mediawiki/2016/3/35/T--USP_UNIFESP-Brazil--mCherry_placaS5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
 +
<img src="https://static.igem.org/mediawiki/2016/d/da/T--USP_UNIFESP-Brazil--mCherry_s10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" /> <br>
 +
&nbsp; &nbsp; &nbsp; &nbsp;Figure 6: pJP22 mCherry transformants - Z5 (TAP Transformed) &nbsp;  &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Figure 7: pJP22 mCherry transformants - Z10 (TAP Transformed)
 +
<img src="https://static.igem.org/mediawiki/2016/e/e7/T--USP_UNIFESP-Brazil--mCherry_placaT5.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" />
 +
<img src="https://static.igem.org/mediawiki/2016/5/5a/T--USP_UNIFESP-Brazil--mCherry_placaT10.jpeg" width= "400px" style="margin-bottom:20px; margin-top:0px;" /> <br>
 +
pJP22 Figure 8: mCherry transformants - Z5 (Sapphire Transformed) &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Figure 9: pJP22 mCherry transformants - Z10 (Sapphire Transformed)<br><br>
 +
<p>As expected, the number of colony-forming units is much higher in the media with less Zeomicin. At the same time, the transformation of cells were not so good using Sapphire Buffer in comparison with Water and TAP medium.
 +
After 7 days we selected clones from previous dishes and started new cultures in a 96 well plate with 200 uL of TAP medium per well, agitation of 800 rpm, 25°C +- 1°C and 80 μE m−2 s−1 luminosity and a clear film sealing the plate. We also filled some wells with wild C. reinhardtii, just TAP medium or just mCherry as controls.</p>
 +
<br><img src="https://static.igem.org/mediawiki/2016/8/88/T--USP_UNIFESP-Brazil--mCherry_screening.png" width=600px><br>
 +
Figure 10: Cultivation setup for screening<br>
 +
<p>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.
 +
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.</p>
 +
The TOP 5 mCherry-producer clones were E 10, which was transformed with TAP medium, and B1, B5, B6 and B11, which were transformed with Sapphire Blue as can be seen in Figure 11.</p>
 +
<img src="https://static.igem.org/mediawiki/2016/5/5c/T--USP_UNIFESP-Brazil--mCherry_top5screening1.png" width=400px><br>
 +
Figure 11: TOP 5 mCherry producers
 +
<p>Before purifying we wanted to see by our own eyes that mCherry was present in the solution. For that, we made the following qualitative analysis:</p>
 +
<img src="https://static.igem.org/mediawiki/2016/2/23/T--USP_UNIFESP-Brazil--mCherry_lasersetup.png" width=400px>
 +
<img src="https://static.igem.org/mediawiki/2016/1/1a/T--USP_UNIFESP-Brazil--mCherry_detection.png " width=400px><br>
 +
Figure 13: Experimental setup for qualitative detection of mCherry.<br>
 +
<p>This special filter is able to block the laser light and at the same time allows the light emitted by mCherry to pass through it, as shown in Figure 14.</p>
 +
<img src="https://static.igem.org/mediawiki/2016/3/39/T--USP_UNIFESP-Brazil--mCherry_superposition.png" width=800px><br>
 +
Image 14: Spectra of experimental setup components
 +
<p>Our results are shown below in Figures 15</p>
 +
<img src="https://static.igem.org/mediawiki/2016/b/be/T--USP_UNIFESP-Brazil--mCherry_lasercontrol.jpeg" width=400px>
 +
<img src="https://static.igem.org/mediawiki/2016/4/48/T--USP_UNIFESP-Brazil--mCherry_lasermcherry.jpeg" width=400px>
 +
<p>Figure 15: Laser passing through cellular supernatant. On the left laser is passing through a wild type C. reinhardtii supernatant. On the right it's passing through a transformed C. reinhardtii producing mCherry.</p>

Revision as of 04:01, 19 October 2016

Description

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:

Before Optimization After Optimization
ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCTTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAATAA ATGGTGTCCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGAGCCCCCAGTTCATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACAGCAGCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCAGCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCAGCAGCGAGCGCATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGCGCCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTGAACATCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGCGCTGAGGGCCGCCACAGCACCGGCGGCATGGACGAGCTGTACAAGTAA
We successfully amplified the codon optimized mCherry, which has 711 bp, as can be seen in image 1.

Image 3 - : Gel electrophoresis of mCherry+pSB1C3 construct

Image 1: Gel electrophoresis of mCherry

After ligating the sequence in the pJP22, we wanted to find the best way to transform C. reinhardtii, we tested Sapphire Blue, TAP medium and water as buffers during the electroporation. Then, cells were plated in Agar-TAP medium Petri dishes with different concentrations of Zeocin (an antibiotic from Bleomycin family) because the plasmid used had a sequence for Bleomycin resistance.


           Figure 2: pJP22 mCherry transformants - Zeocin 5 ug/mL                    Figure 3: pJP22 mCherry transformants - Zeocin 10 ug/mL

        Figure 4: pJP22 mCherry transformants - Z5 (Water Transformed)                Figure 5: pJP22 mCherry transformants - Z10 (Water Transformed)
       Figure 6: pJP22 mCherry transformants - Z5 (TAP Transformed)               Figure 7: pJP22 mCherry transformants - Z10 (TAP Transformed)
pJP22 Figure 8: mCherry transformants - Z5 (Sapphire Transformed)                  Figure 9: pJP22 mCherry transformants - Z10 (Sapphire Transformed)

As expected, the number of colony-forming units is much higher in the media with less Zeomicin. At the same time, the transformation of cells were not so good using Sapphire Buffer in comparison with Water and TAP medium. After 7 days we selected clones from previous dishes and started new cultures in a 96 well plate with 200 uL of TAP medium per well, agitation of 800 rpm, 25°C +- 1°C and 80 μE m−2 s−1 luminosity and a clear film sealing the plate. We also filled some wells with wild C. reinhardtii, just TAP medium or just mCherry as controls.



Figure 10: Cultivation setup for screening

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. 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.

The TOP 5 mCherry-producer clones were E 10, which was transformed with TAP medium, and B1, B5, B6 and B11, which were transformed with Sapphire Blue as can be seen in Figure 11.


Figure 11: TOP 5 mCherry producers

Before purifying we wanted to see by our own eyes that mCherry was present in the solution. For that, we made the following qualitative analysis:


Figure 13: Experimental setup for qualitative detection of mCherry.

This special filter is able to block the laser light and at the same time allows the light emitted by mCherry to pass through it, as shown in Figure 14.


Image 14: Spectra of experimental setup components

Our results are shown below in Figures 15

Figure 15: Laser passing through cellular supernatant. On the left laser is passing through a wild type C. reinhardtii supernatant. On the right it's passing through a transformed C. reinhardtii producing mCherry.