Difference between revisions of "Team:Humboldt Berlin/Contribution"

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             <h1 class="page-headline description" style="font-size:140px;color:#ffffff">Characterization</h1>
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We encountered problems when measuring the the YFP-signal in our transformed <i>C. rheinhardtii</i> YFP strains, even though we were able to detect YFP when examining the cells with the fluorescence microscope. To get a better understanding of the protocols followed to measure absorbance and fluorescence spectra with a plate reader, we conducted some exemplary measurements with fluorescent and non-fluorescent chromoproteins expressed in <i>E. coli</i>.  
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We encountered problems when measuring the YFP-signal in our <i>C. reinhardtii</i> strains transformed with YFP, even though we were able to detect YFP when examining the cells with the fluorescence microscope. To get a better understanding of the protocols followed to measure absorbance and fluorescence spectra with a plate reader, as an example we conducted some measurements with fluorescent and non-fluorescent chromoproteins expressed in <i>E. coli</i>.  
 
We tested three different chromoproteins from the iGEM Registry of Standard Biological Parts in terms of their absorption spectra (as well as the fluorescence spectrum in case of amajLime): <br/></p>
 
We tested three different chromoproteins from the iGEM Registry of Standard Biological Parts in terms of their absorption spectra (as well as the fluorescence spectrum in case of amajLime): <br/></p>
 
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<li>spisPink from the coral <i>Stylophora pistillata</i> <a href="http://parts.igem.org/Part:BBa_K1033923">BBa_K1033923</a></li>
 
<li>spisPink from the coral <i>Stylophora pistillata</i> <a href="http://parts.igem.org/Part:BBa_K1033923">BBa_K1033923</a></li>
 
<li>gfasPurple from the coral <i>Galaxea fascicularis</i>  <a href="http://parts.igem.org/Part:BBa_K1033917">BBa_K1033917</a> </li>
 
<li>gfasPurple from the coral <i>Galaxea fascicularis</i>  <a href="http://parts.igem.org/Part:BBa_K1033917">BBa_K1033917</a> </li>
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</ul><p class="block-text medium-sized">Therefore we transformed the pSB1C3 vector with the respective chromoprotein constructs (which all carry a <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23110">promotor </a>  and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">RBS sequence</a>) into E. coli (DH10B Competent Cells). After cultivation and we lysed the harvested cells according to our <a href="https://2019.igem.org/Team:Humboldt_Berlin/Experiments"> lysis protocol </a> .
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</ul><p class="block-text medium-sized">Therefore we transformed the pSB1C3 vector with the respective chromoprotein construct (which all carry a <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23110">promotor </a>  and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">RBS sequence</a>) into <i>E. coli</i> (DH10B Competent Cells). After cultivation we lysed the harvested cells according to our <a href="https://2019.igem.org/Team:Humboldt_Berlin/Experiments"> lysis protocol.</a>
  
 
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With a plate reader (TECAN Plate Reader Infinite 200 Pro) we measured the absorbance (and fluorescence) spectra of the cleared lysate. For the interpretation of the spectra we utilized our lysis buffer as blank measurement. 
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We obtained characteristic absorbance spectra for all three chromoproteins, with the respective absorbance peak equivalent to the values described in the literatur (Liljeruhm et al. 2018). For the fluorescent chromoprotein amajLime we were able to measure a evident fluorescence spectrum with a clear peak at xxx nm.
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                         <img class="is-revealing" src="https://static.igem.org/mediawiki/parts/3/32/Humboldt_Berlin_Contr_chromo-pellet-pink-purple.jpg" alt="Chromoproteins pink & purple" />
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                         <figure class="is-revealing" > <img src="https://static.igem.org/mediawiki/parts/3/32/Humboldt_Berlin_Contr_chromo-pellet-pink-purple.jpg" alt="Chromoproteins spisPink & gfasPurple" /><figcaption> Fig. 1. - Picture of cell pellets: gfasPurple & spisPink</figcaption>
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                        <img class="is-revealing" src="https://static.igem.org/mediawiki/parts/e/e4/Humboldt_Berlin_Contr_chromo-pellet-lime-controlle.jpg" alt="Chromoproteins lime & control" />
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                      <figure class="is-revealing" >  <img src="https://static.igem.org/mediawiki/parts/e/e4/Humboldt_Berlin_Contr_chromo-pellet-lime-controlle.jpg" alt="Chromoproteins amajLime & control" /><figcaption> Fig. 2. - Picture of cell pellets: amajLime & non-colored control</figcaption>
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With a plate reader (TECAN Plate Reader Infinite 200 Pro) we measured the absorbance (and fluorescence) spectra of the cleared lysate (Figure 3). For the interpretation of the spectra we utilized our lysis buffer as a blank measurement. 
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We obtained characteristic absorbance spectra for all three chromoproteins (Figure 4-6), with the respective absorbance peak equivalent to the values previously described in the literature (Liljeruhm et al. 2018). For the fluorescent chromoprotein amajLime we were able to measure an evident fluorescence spectrum with a clear peak at 493 nm (Figure 7). All spectra are plotted including their respective standard deviations. In Figure 8 the relative absorbance and fluorescence spectra of amajLime are shown to emphasize the Stokes shift of the spectral peaks. 
  
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                            <figcaption>Fig.3. - Picture of lysate amajLime, gfasPurple, spisPink, control: </figcaption>
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    <th colspan="2" scope="col" style="padding-left: 15px">Table 1. Parameters utilized for the measurement of absorbance and fluorescence spectra</th>
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                                                      <th scope="col" style="padding-left: 15px">Parameter</th>
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                                                        <th scope="col">Value</th>
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                                                      <td style="padding-left: 15px">Number of Samples </td>
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                                                        <td>24 </td>
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                                                      <td style="padding-left: 15px">Wavelength Step Size </td>
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                                                        <td>2 </td>
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                                                      <td style="padding-left: 15px">Absorbance Scan: Excitation Wavelength Measurement Range (nm)</td>
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                                                        <td>[300-800]</td>
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                                                      <td style="padding-left: 15px">Fluorescence Scan: Emission Wavelength Measurement Range (nm)</td>
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                                                        <td>[475-550] </td>
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                                                      <td style="padding-left: 15px">Fluorescence Scan: Excitation Wavelength (nm)</td>
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                                                        <td>445 </td>
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                                                        <td>52</td>
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                                                        <td>25</td>
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                                                        <td>20</td>
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                                                        <td>0</td>
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                                                        <td>0</td>
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                         <figure class="is-revealing" > <img src="https://static.igem.org/mediawiki/parts/7/7f/T--Humboldt_Berlin--CharacterisationGfasPurpleAbs.png" alt="Absorbance gfasPurple" /><figcaption> Fig. 4. - Absorbance spectrum of gfasPurple</figcaption>
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                      <figure class="is-revealing" >  <img src="https://static.igem.org/mediawiki/parts/e/ec/T--Humboldt_Berlin--CharacterisationspisPinkAbs.png" alt="Absorbance spisPink" /><figcaption> Fig. 5. - Absorbance spectrum of spisPink</figcaption>
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                        <figure class="is-revealing" > <img src="https://static.igem.org/mediawiki/parts/c/ce/T--Humboldt_Berlin--CharacterisationAmajLimeAbs.png" alt="Absorbance amajLime" /><figcaption> Fig. 6. - Absorbance spectrum of amajLime </figcaption>
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                      <figure class="is-revealing" > <img src="https://static.igem.org/mediawiki/parts/0/05/T--Humboldt_Berlin--CharacterisationAmajLimeFluo.png" alt="" /><figcaption> Fig. 7. -  Fluorescence spectrum of amajLime </figcaption>
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absorbance and fluorescence spectra of amajLime</figcaption>
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Latest revision as of 12:23, 18 October 2019

Colorporteins

Characterization

It's colorful

Characterizing Chromoproteins

We encountered problems when measuring the YFP-signal in our C. reinhardtii strains transformed with YFP, even though we were able to detect YFP when examining the cells with the fluorescence microscope. To get a better understanding of the protocols followed to measure absorbance and fluorescence spectra with a plate reader, as an example we conducted some measurements with fluorescent and non-fluorescent chromoproteins expressed in E. coli. We tested three different chromoproteins from the iGEM Registry of Standard Biological Parts in terms of their absorption spectra (as well as the fluorescence spectrum in case of amajLime):

Therefore we transformed the pSB1C3 vector with the respective chromoprotein construct (which all carry a promotor and RBS sequence) into E. coli (DH10B Competent Cells). After cultivation we lysed the harvested cells according to our lysis protocol.

Picture Chromoproteins
Chromoproteins spisPink & gfasPurple
Fig. 1. - Picture of cell pellets: gfasPurple & spisPink
Chromoproteins amajLime & control
Fig. 2. - Picture of cell pellets: amajLime & non-colored control

With a plate reader (TECAN Plate Reader Infinite 200 Pro) we measured the absorbance (and fluorescence) spectra of the cleared lysate (Figure 3). For the interpretation of the spectra we utilized our lysis buffer as a blank measurement. We obtained characteristic absorbance spectra for all three chromoproteins (Figure 4-6), with the respective absorbance peak equivalent to the values previously described in the literature (Liljeruhm et al. 2018). For the fluorescent chromoprotein amajLime we were able to measure an evident fluorescence spectrum with a clear peak at 493 nm (Figure 7). All spectra are plotted including their respective standard deviations. In Figure 8 the relative absorbance and fluorescence spectra of amajLime are shown to emphasize the Stokes shift of the spectral peaks.

´Tubes with Cromoproteins
Fig.3. - Picture of lysate amajLime, gfasPurple, spisPink, control:
Table 1. Parameters utilized for the measurement of absorbance and fluorescence spectra
Parameter Value
Number of Samples 24
Wavelength Step Size 2
Absorbance Scan: Excitation Wavelength Measurement Range (nm) [300-800]
Fluorescence Scan: Emission Wavelength Measurement Range (nm) [475-550]
Fluorescence Scan: Excitation Wavelength (nm) 445
Gain (G) 52
Number of Flashes 25
Integration Time (µs) 20
Lag Time (µs) 0
Settle Time (ms) 0
Absorbance gfasPurple
Fig. 4. - Absorbance spectrum of gfasPurple
Absorbance spisPink
Fig. 5. - Absorbance spectrum of spisPink
Absorbance amajLime
Fig. 6. - Absorbance spectrum of amajLime
Fig. 7. - Fluorescence spectrum of amajLime
Absorbance amajLime
Fig. 8. - Relative absorbance and fluorescence spectra of amajLime

Liljeruhm, Josefine et al. “Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology.” Journal of biological engineering vol. 12:8. 10 May. 2018, doi:10.1186/s13036-018-0100-0