Difference between revisions of "Team:DTU-Denmark/Results-melA"

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<p>Here you can describe the results of your project and your future plans. </p>
 
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<h3>What should this page contain?</h3>
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<li> Clearly and objectively describe the results of your work.</li>
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<li> Future plans for the project. </li>
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<li> Considerations for replicating the experiments. </li>
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<i>melA</i>, from <i>Rhizobium etli</i> encodes a tyrosinase involved in the first step of the melanin synthesis, producing dopaquinone. To increase the resistance of our fungi to UV radiation we were interested in increasing expression of melanin, as the pigment has previously been reported to protect against UV radiation. (1)
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<h2 class="media-heading" style="text-align: left;margin-bottom: 35px; color:#50C8E8;">Assembly</h2>
  
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<h3>Describe what your results mean </h3>
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
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<i>melA</i> was synthesized <i>de novo</i> by IDT and assembled with <i>pTrpC</i> into the pSB1C3 backbone by 3A assembly. The cauliflower mosaic virus terminator was then added by 3A assembly. Correct transformants were identified by colony PCR and Sanger sequencing using the verification primers, VF2 and VR (data not shown). The plasmid map of the biobrick can be seen in figure 1:
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<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/c/ca/T--DTU-Denmark--results_melA_BBa_K2799013.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 1: </b> -Plasmid map of BBa_K2799013</p></figcaption>
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<h2 class="media-heading"  style="text-align: right;margin-bottom: 35px; color:#F8A05B;">Transformation</h2>
  
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<i>A. oryzae</i> protoplasts were transformed with the <i>melA</i> coding biobrick BBa_K2799013 and plated on transformation media w. 0.6 mg/µL L-tyrosine. After ~3 days of growth almost all colonies exhibited wild type phenotypic behaviour and morphology, which is to be expected as there is no selectable marker for successful transformants.
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However, one area of colony exhibiting darker color than the surrounding colonies might be indicative of a successful transformation. We have replated the colony to further assess whether the potential transformant exhibits consistent darker color, in which case we will validate the insertion of our biobrick by PCR and subsequently sequencing.
  
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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/4/49/T--DTU-Denmark--results_melA_possible_transformant.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 2: </b> - Plate with colony of interest from plating of melA transformants.
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<h3>Inspiration</h3>
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<h2 class="media-heading" style="text-align: left;margin-bottom: 35px; color:#50C8E8;">Brick construction</h2>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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To test whether the integration, and possible expression of <i>amilCP</i>, would lead to a visual change in materials produced, a small brick was inoculated using spores from the identified transformants. However, this did not produce any visual obvious change in brick color (data not shown).
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<h2 class="media-heading"  style="text-align: right;margin-bottom: 35px; color:#F8A05B;">Conclusion</h2>
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A slight visual difference in spore colouration can be observed in possible transformants corresponding (see figure 4) to integration of the expression cassette as demonstrated by PCR (see figure 5 and 6). This indicates production of AmilCP as it’s a blue chromoprotein. However, to definitely demonstrate this further test are needed. Given more time we would like to have demonstrated by qPCR as well as show the actual presences of AmilCP in the transformants by mass spectrometry.
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In order to change the color of the bricks produced from it higher expression seems to be necessary. This could feasibly be by codon-optimizing the sequence for expression in <i>A. oryzae</i>
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<a href="https://static.igem.org/mediawiki/2018/3/3e/T--DTU-Denmark--Primer_list.pdf">Primer list</a>
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<p style="color:#000; font-size:14px;">(1) Jing, G.; Sheng-Bing, Y.; Xia, W.; Xiao-Juan, W.; Ping, S., Ping, Z., Xiang-Dong, C. Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system. Journal of biochemical and biophysical methods. <i>Elsevier</i>, 70(6), 2008, 70, 1151-1155. doi: 10.1016/J.JPROT.2007.12.013.
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Revision as of 00:17, 18 October 2018

melA Expression

melA, from Rhizobium etli encodes a tyrosinase involved in the first step of the melanin synthesis, producing dopaquinone. To increase the resistance of our fungi to UV radiation we were interested in increasing expression of melanin, as the pigment has previously been reported to protect against UV radiation. (1)

Assembly

melA was synthesized de novo by IDT and assembled with pTrpC into the pSB1C3 backbone by 3A assembly. The cauliflower mosaic virus terminator was then added by 3A assembly. Correct transformants were identified by colony PCR and Sanger sequencing using the verification primers, VF2 and VR (data not shown). The plasmid map of the biobrick can be seen in figure 1:

Fig. 1: -Plasmid map of BBa_K2799013

Transformation

A. oryzae protoplasts were transformed with the melA coding biobrick BBa_K2799013 and plated on transformation media w. 0.6 mg/µL L-tyrosine. After ~3 days of growth almost all colonies exhibited wild type phenotypic behaviour and morphology, which is to be expected as there is no selectable marker for successful transformants. However, one area of colony exhibiting darker color than the surrounding colonies might be indicative of a successful transformation. We have replated the colony to further assess whether the potential transformant exhibits consistent darker color, in which case we will validate the insertion of our biobrick by PCR and subsequently sequencing.

Fig. 2: - Plate with colony of interest from plating of melA transformants.

Brick construction

To test whether the integration, and possible expression of amilCP, would lead to a visual change in materials produced, a small brick was inoculated using spores from the identified transformants. However, this did not produce any visual obvious change in brick color (data not shown).

Conclusion

A slight visual difference in spore colouration can be observed in possible transformants corresponding (see figure 4) to integration of the expression cassette as demonstrated by PCR (see figure 5 and 6). This indicates production of AmilCP as it’s a blue chromoprotein. However, to definitely demonstrate this further test are needed. Given more time we would like to have demonstrated by qPCR as well as show the actual presences of AmilCP in the transformants by mass spectrometry. In order to change the color of the bricks produced from it higher expression seems to be necessary. This could feasibly be by codon-optimizing the sequence for expression in A. oryzae

Primer list

(1) Jing, G.; Sheng-Bing, Y.; Xia, W.; Xiao-Juan, W.; Ping, S., Ping, Z., Xiang-Dong, C. Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system. Journal of biochemical and biophysical methods. Elsevier, 70(6), 2008, 70, 1151-1155. doi: 10.1016/J.JPROT.2007.12.013.