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

 
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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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The <i>melA</i> gene 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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<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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<i>melA</i> was synthesized <i>de novo</i> by IDT and assembled with P<i>trpC</i> into the pSB1C3 backbone by 3A assembly. The cauliflower mosaic virus (CaMV) 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 fig. 1:
 
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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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<p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/6/6f/T--DTU-Denmark--results_melA_BBa_K2799014.png" style="max-width: 100%;" > <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 1: </b>Plasmid map of BBa_K2799014</p></figcaption>
  
  
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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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<i>A. oryzae</i> protoplasts were transformed with the <i>melA</i> coding biobrick BBa_K2799014 and plated on transformation media w. 0.6 mg/mL 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.
 
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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<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 <i>melA</i> transformants.
 
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<h2 class="media-heading" style="text-align: left;margin-bottom: 35px; color:#50C8E8;">Brick construction</h2>
 
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<h2 class="media-heading"  style="text-align: left;margin-bottom: 35px; color:#50C8E8;">Conclusion</h2>
  
 
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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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We have successfully assembled two <i>melA</i> cassettes, each with a different promoter. <br>
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As of now, we have apparent transformants, which suggests integration of our cassette into the <i>A. oryzae</i> genome. If this can be verified, it opens the opportunity to fabricate fungal materials with the added benefit of melanin inducing UV resistance.
  
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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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<a target="_blank" href="https://static.igem.org/mediawiki/2018/3/3e/T--DTU-Denmark--Primer_list.pdf">Appendix: Primer list</a>
 
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<a href="https://2018.igem.org/Team:DTU-Denmark/Description">Project description</a>
 
<a href="https://2018.igem.org/Team:DTU-Denmark/Description">Project description</a>
 
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                       <a href="https://2018.igem.org/Team:DTU-Denmark/Model">Modelling</a>
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                       <a href="https://2018.igem.org/Team:DTU-Denmark/Model">Modeling</a>
 
&bull;
 
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<a href="https://2018.igem.org/Team:DTU-Denmark/Parts">Parts overview</a>
 
<a href="https://2018.igem.org/Team:DTU-Denmark/Parts">Parts overview</a>

Latest revision as of 02:54, 18 October 2018

melA Expression

The melA gene 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 (CaMV) 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 fig. 1:

Fig. 1: Plasmid map of BBa_K2799014

Transformation

A. oryzae protoplasts were transformed with the melA coding biobrick BBa_K2799014 and plated on transformation media w. 0.6 mg/mL 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.

Conclusion

We have successfully assembled two melA cassettes, each with a different promoter.
As of now, we have apparent transformants, which suggests integration of our cassette into the A. oryzae genome. If this can be verified, it opens the opportunity to fabricate fungal materials with the added benefit of melanin inducing UV resistance.

Appendix: 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.