Difference between revisions of "Part:BBa K2984000"
 
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This vector is a part of the <a href="https://2019.igem.org/Team:Humboldt_Berlin/Part_Collection">Chlamy-HUB-Collection</a>. For the secretion of a protein in <i>Chlamydomonas reinhardtii</i> it is necessary that this protein is attached to a signal sequence that targets it to the secretory pathway and eventually into the medium. It has been demonstrated that the Arylsulfatase 1 (ARS1) is not only able to direct protein synthesis to the secretory pathway, it was also shown that it is able to secrete heterologous proteins (Rasala et al., 2012). The amino acid sequence of the signal peptide of ARS1 is MHARKMGALAVLAVACLAAVASVAHA.
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This vector is a part of the <a href="https://2019.igem.org/Team:Humboldt_Berlin/Part_Collection">Chlamy-HUB-Collection</a>. For the secretion of a protein in <i>Chlamydomonas reinhardtii</i> it is necessary that this protein is attached to a signal sequence that targets it to the secretory pathway and eventually into the medium. It has been demonstrated that the Arylsulfatase 1 (ARS1) is not only able to direct protein synthesis to the secretory pathway, it was also shown that it is able to secrete heterologous proteins (Rasala et al., 2012). The amino acid sequence of the signal peptide of ARS1 is MHARKMGALAVLAVACLAAVASVAHA.  
 
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This part was designed to be used with the MoClo standard and has B2-B2 overhangs.
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===Usage and Biology===
 
===Usage and Biology===
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2984000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2984000 SequenceAndFeatures</partinfo>
  
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==Characterization==
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<figure>
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<img src="https://static.igem.org/mediawiki/parts/8/8c/T--Humboldt_Berlin--ARS1.png" alt="Platte_ARS" width="500">
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<figcaption>Fig.1 - Successful transformation of ARS into <i>E. coli</i>.</figcaption>
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<p>
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We were able to demonstrate that in a construct where SP<sub>20</sub> was used (<a href="https://parts.igem.org/Part:BBa_K2984030">BBa_K2984030</a>), vesicles containing YFP mVEnus were formed (Fig .1)
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</p>
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<img class="is-revealing" src="https://2019.igem.org/wiki/images/b/b2/T--Humboldt_Berlin--Microscopy-YFP.png" alt="fluorescence difference spectrum" width="600" />
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<figcaption>Fig. 1 - Presence of YFP mVenus in <i>C. reinhardtii</i>. For construct BBa K2984030 it can be seen that a vesicle with a high concentration of YFP mVenus was formed near the membrane. Taken with a confocal microscope.</figcaption>
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=Blue & Green Screening=
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The effect of the arylsulfatase secretion signal is used for screening of postive Cas9 incision in the SNRK locus in <i>C.reinhardtii</i>. The target site lies within a regultory unit, inhibiting the expression of the arylsulfatse. When cut, expression of the enzyme rises. Outside the cell the acitivity of the enzyme turns a supplemented color into blue proving the functionallity of the signal.
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<figure>
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<img src="https://2019.igem.org/wiki/images/f/f0/T--Humboldt_Berlin--WhenARSworks.jpeg" alt="colonies_total" width="500">
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<figcaption>Fig.3 - <i>C. reinahrdtii </i> colonies on TAP-Agar plate. The blue colonies indicate a successfull incision inside the SNRK locus which regulates the expression of the arylsulfatase. The blue color is generated through the activity of the arylsulfatase outside the cells, verifiying the functionality of the ARS secretion signal.</figcaption>
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<partinfo>BBa_K2984000 parameters</partinfo>
 
<partinfo>BBa_K2984000 parameters</partinfo>
 
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==References==
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<ol>
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Rasala, B. A., Lee, P. A., Shen, Z., Briggs, S. P., Mendez, M., & Mayfield, S. P. (2012). Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PloS one, 7(8), e43349.
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</li>
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</ol>

Latest revision as of 21:52, 21 October 2019


3'UTR, Secretion signal of Arylsulfatase ARS B2-B2

This vector is a part of the Chlamy-HUB-Collection. For the secretion of a protein in Chlamydomonas reinhardtii it is necessary that this protein is attached to a signal sequence that targets it to the secretory pathway and eventually into the medium. It has been demonstrated that the Arylsulfatase 1 (ARS1) is not only able to direct protein synthesis to the secretory pathway, it was also shown that it is able to secrete heterologous proteins (Rasala et al., 2012). The amino acid sequence of the signal peptide of ARS1 is MHARKMGALAVLAVACLAAVASVAHA.

This part was designed to be used with the MoClo standard and has B2-B2 overhangs.


Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Platte_ARS
Fig.1 - Successful transformation of ARS into E. coli.

We were able to demonstrate that in a construct where SP20 was used (BBa_K2984030), vesicles containing YFP mVEnus were formed (Fig .1)

fluorescence difference spectrum
Fig. 1 - Presence of YFP mVenus in C. reinhardtii. For construct BBa K2984030 it can be seen that a vesicle with a high concentration of YFP mVenus was formed near the membrane. Taken with a confocal microscope.


Blue & Green Screening

The effect of the arylsulfatase secretion signal is used for screening of postive Cas9 incision in the SNRK locus in C.reinhardtii. The target site lies within a regultory unit, inhibiting the expression of the arylsulfatse. When cut, expression of the enzyme rises. Outside the cell the acitivity of the enzyme turns a supplemented color into blue proving the functionallity of the signal.

colonies_total
Fig.3 - C. reinahrdtii colonies on TAP-Agar plate. The blue colonies indicate a successfull incision inside the SNRK locus which regulates the expression of the arylsulfatase. The blue color is generated through the activity of the arylsulfatase outside the cells, verifiying the functionality of the ARS secretion signal.



References

  1. Rasala, B. A., Lee, P. A., Shen, Z., Briggs, S. P., Mendez, M., & Mayfield, S. P. (2012). Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PloS one, 7(8), e43349.