Difference between revisions of "Part:BBa K1769009"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1769009 short</partinfo>
 
<partinfo>BBa_K1769009 short</partinfo>
  
−
123
 
  
−
<!-- Add more about the biology of this part here
+
 
 +
 
 
===Usage and Biology===
 
===Usage and Biology===
 +
<html>
 +
<p>Most fungicide used to cure or prevent potato late blight contain heavy metal, such as copper. these heavy metal will not only kill p.infestans, a pathogen that causes late blight, but also do harm to the plant and environment. Therefore, we created a defensin called lm-def. It is a antifungal peptide from Lepidium meyenii, an Andean crop, that can effectively and specifically inhibit the growth of p.infestans zoospores and destroy the hyphae of p.infestans. </p>
 +
</html>
 +
==Antimicrobial assay==
 +
<html>
 +
<p>To test if zoospores and hyphae of p.infestans will be destroyed by our defensin lm-def. We carry out an antimicrobial assay experiment. we incubated in advance the p.infestans on rye agar plates at 18 degrees Celsius for ten days to acquire sporangia and hyphal pieces. Dilution broth solution was then prepared by boiling fresh pea seeds for 20 minutes in distilled water and autoclaved for sterilization. We harvested P. infestans in sterile water; later, we used hemocytometer to count the number of sporangia and diluted it to 250 sporangia per 100 microliter with the dilution broth solution. We took the 100 ul spore suspension and added to it 50 ul of recombinant protein in a 96 well microtiter plate. We used PBS as blank, the pET29b empty vector as protein control, and the spore suspension for comparison. The mixture will then be incubated in darkness at 18 degrees Celsius for an entire day. Spore germination will be measured with a plate reader at 595 nm at the beginning of the test and 24 hours after inoculation. Apart from that, we monitored changes in hyphal growth by physically cutting out agar and placing it in a 12-well tissue culture plate with each well loaded with 1600 ul of lysate while using dilution broth solution as positive control, as it theoretically did not contain any substance that would suppress the growth of the oomycete. We inspected the samples sliced out of the agar every two hours under a light microscope.
 +
</p>
 +
</html>
 +
==Results==
 +
<html>
 +
<style>
 +
 +
 +
div.item {
 +
    /* To correctly align image, regardless of content height: */
 +
    vertical-align: top;
 +
    display: inline-block;
 +
    /* To horizontally center images and caption */
 +
    text-align: center;
 +
    /* The width of the container also implies margin around the images. */
 +
    width: 24%;
 +
 +
}
 +
 +
.caption {
 +
    /* Make the caption a block so it occupies its own line. */
 +
    display: block;
 +
    font-weight:bold;
 +
    font-size:16px;
 +
}
 +
</style>
 +
<p>We observe the morphology of P.infestans under microscopy every 2 hours after treating zoospores with lm-def. The picture bellow shows the differences between the morphology begore and after treatment.</p><br>
 +
<div class="item" style="padding-left:16%;width:22%">
 +
<img src="https://static.igem.org/mediawiki/parts/4/4e/Before_treat.jpg" style="padding-left:15%;max-width:40%; display:inline-block;">
 +
<span class="caption">Hyphae of p.infestans before treatment</span>
 +
</div>
 +
<div class="item" style="padding-left:16%;width:22%">
 +
<img src="https://static.igem.org/mediawiki/parts/6/65/Treat.jpg" style="max-width:40%;display:inline-block;">
 +
<span class="caption">Hyphae of p.infestans after treatment</span>
 +
</div>
 +
  
−
<!-- -->
+
<p>>Sequence and Features</p>
−
<span class='h3bb'>Sequence and Features</span>
+
</html>
 
<partinfo>BBa_K1769009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1769009 SequenceAndFeatures</partinfo>
  

Revision as of 11:35, 18 September 2015

T7+RBS+Lm-def



Usage and Biology

Most fungicide used to cure or prevent potato late blight contain heavy metal, such as copper. these heavy metal will not only kill p.infestans, a pathogen that causes late blight, but also do harm to the plant and environment. Therefore, we created a defensin called lm-def. It is a antifungal peptide from Lepidium meyenii, an Andean crop, that can effectively and specifically inhibit the growth of p.infestans zoospores and destroy the hyphae of p.infestans.

Antimicrobial assay

To test if zoospores and hyphae of p.infestans will be destroyed by our defensin lm-def. We carry out an antimicrobial assay experiment. we incubated in advance the p.infestans on rye agar plates at 18 degrees Celsius for ten days to acquire sporangia and hyphal pieces. Dilution broth solution was then prepared by boiling fresh pea seeds for 20 minutes in distilled water and autoclaved for sterilization. We harvested P. infestans in sterile water; later, we used hemocytometer to count the number of sporangia and diluted it to 250 sporangia per 100 microliter with the dilution broth solution. We took the 100 ul spore suspension and added to it 50 ul of recombinant protein in a 96 well microtiter plate. We used PBS as blank, the pET29b empty vector as protein control, and the spore suspension for comparison. The mixture will then be incubated in darkness at 18 degrees Celsius for an entire day. Spore germination will be measured with a plate reader at 595 nm at the beginning of the test and 24 hours after inoculation. Apart from that, we monitored changes in hyphal growth by physically cutting out agar and placing it in a 12-well tissue culture plate with each well loaded with 1600 ul of lysate while using dilution broth solution as positive control, as it theoretically did not contain any substance that would suppress the growth of the oomycete. We inspected the samples sliced out of the agar every two hours under a light microscope.

Results

We observe the morphology of P.infestans under microscopy every 2 hours after treating zoospores with lm-def. The picture bellow shows the differences between the morphology begore and after treatment.


Hyphae of p.infestans before treatment
Hyphae of p.infestans after treatment

>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]