Difference between revisions of "Team:NYMU-Taipei/Project-Experiment"

 
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<h2 style="margin-top:30px; margin-bottom:10px;">Selection markers</h2><hr>
  
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<p style="font-size:16px;">Firstly we tested for effective fungal selection marker <i>hph</i> and <i>ble</i> (Corresponding  antibiotics: hygromycin and phleomycin)
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<p style="font-size:16px;"> The concentration for hygromycin selection ranges from 200-1000 µg/ml<sup>[1]</sup> and phleomycin-ranges from 10-50 µg/ml <sup>[2]</sup> for fungi.
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We chose this series of antibiotic concentration:
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<p style="font-size:16px;">    Hygromycin (μg/mL): 0, 50, 100, 150, 200
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<p style="font-size:16px;"> Phleomycin (μg/mL): 0, 25, 50
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<i>M. anisopliae</i> were incubated in each of the antibiotics test plates and incubated at 25°C.
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<p style="font-size:16px;">Fig.1 Hygromycin test plates (1-5: 0, 50, 100, 150, 200 ug/mL)  
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<p style="font-size:16px;">Fig.2 Phleomycin test plates (1-3: 0, 25, 50 ug/mL)  
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<p style="font-size:16px;">As the images shown above, the growing situation of wild type strain <i>M. anisopliae</i> on hygromycin or phleomycin plates are nearly the same comparing to the control. It indicated that our chassis fungi <i>Metarhizium anisopliae</i> ARSEF549 is not sensitive to hygromycin and phleomycin, that means hygromycin and phleomycin can not select transformants during transformation.
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<h2 style="margin-top:30px; margin-bottom:10px;">Insect hemolymph bioassays</h2><hr>
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    We extracted insect hemolymph from three different species: oriental fruit flies, cherry cockroaches and silkworms.
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<p style="font-size:16px;">Fig.3 Oriental fruit flies
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<p style="font-size:16px;">Fig.4 Extracting cherry cockroach's hemolymph
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<p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p><p>menu</p>
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<img src="https://static.igem.org/mediawiki/parts/f/f7/Silkworm.jpeg" height="250" width="250">
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<p style="font-size:16px;">Fig.5 Silkworms
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</p>
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<p style="font-size:16px;">
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After 24 hours(in cherry cockroach and silkworm's hemolymph) and 30 hours(in oriental fruit fly's hemolymph) cultivation, the fungal cells were observed using the bright field microscopy(Magnification: 1000X).
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</p>
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<div id="DIV2">
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<img src="https://static.igem.org/mediawiki/parts/e/e8/Fly_done.png" height="250" width="250">
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<p style="font-size:16px;">Fig.6 <i>M. anisopliae</i> in oriental fruit fly's hemolymph for 30 hours
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</p>
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</div>
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<div id="DIV2">
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<img src="https://static.igem.org/mediawiki/parts/6/65/Coach_done.png" height="250" width="250">
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<p style="font-size:16px;">Fig.7 <i>M. anisopliae</i> in cherry coach's hemolymph for 24 hours
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</p>
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</div>
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 +
 
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<img src="https://static.igem.org/mediawiki/parts/f/fc/Silkworm_done.png" height="250" width="250">
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<p style="font-size:16px;">Fig.8 <i>M. anisopliae</i> in silkworm's hemolymph for 24 hours
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</p>
 +
 
 +
<p style="font-size:16px;"> Appressorium development can be clearly observed after 24h induction within the hemolymph of silkworms.
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</p>
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</div>
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<h2 style="margin-top:30px; margin-bottom:10px;"><i>Mcl1</i> promoter</h2><hr>
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<p style="font-size:16px;">  
 +
    We designed the primers PMcl1-f(ACGTC//CTGCAG//AATCATGCAGCGCTATGAG, with a PstI site underlined) and PMcl1-r(ATAA//GCGGCCGC//CATGATGGTCTAGGGAACG with a NotI site underlined), according to the <i>PMcl1</i> sequence<sup>[1]</sup>, to amplify the <i>Mcl1</i> promoter region with <i>Mcl1</i> mRNA 5'-untranslated region at the 5' end of the coding region. The whole length is 2772bp.
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</p>
 +
 
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<p style="font-size:16px;">  
 +
    The gel image below shows that we succeed extracting the <i>Mcl1</i> promoter and its 5'-untranslated region (99bp downstream the promoter) from the genomic DNA of our chassis organism <i>M. anisopliae</i> ARSEF549.
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</p>
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<div style="border:1px solid:black">
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<img src="https://static.igem.org/mediawiki/parts/d/de/PMcl1_PCR.png" width="40%">
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<p style="font-size:16px;">Fig.9  Amplify <i>PMcl1</i> from gDNA
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</p>
 +
</div>
 +
 
 +
<p style="font-size:16px;">   Then we digested the DNA fragment with NotI and PstI in order to insert it into the backbone. However, when we ran gel electrophoresis to check the digestion result, we found that there is still one unknown PstI cut site inside the PMcl1 region.
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</p>
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<div style="border:1px solid:black">
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<img src="https://static.igem.org/mediawiki/parts/0/05/PMcl1_digest.jpeg" width="40%">
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<p style="font-size:16px;">Fig.10  The broken PMcl1 fragment
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</p>
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</div>
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<p style="font-size:16px;">  
 +
    We decided to sequence this DNA fragment we extracted and mutate the PstI site, but we didn't have enough time to finish our relative vectors construction.
 +
</p>
 +
 
 +
<h2 style="margin-top:30px; margin-bottom:10px;">KillerRed expression in <i>M. anisopliae</i></h2><hr>
 +
 
 +
<p style="font-size:16px;">   We constructed a KillerRed expression cassette with a fungal promoter <i>PgpdA</i> and a fungal terminator <i>TtrpC</i>. This cassette was used to confirm that KillerRed can be expressed in <i>M. anisopliae</i>
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/2016/0/02/T-NYMU-Taipei-photo-PMcl1_KR_TtrpC1.jpeg" width="60%">
 +
 
 +
<p style="font-size:16px;">   *The fluorescence images below indicated that KillerRed protein was successfully expressed in <i>M. anisopliae</i>.
 +
</p>
 +
<img src="https://static.igem.org/mediawiki/parts/0/0f/PKT_FL.jpeg" width="90%">
 +
<p style="font-size:16px;">   As we observed, the growth situations of <i>M. anisopliae</i> KR transformants on media will not be affected greatly since irradiation of KillerRed localized in cell cytosol has a weak effect on cell survival in eukaryotic cells<sup>[3]</sup>.
 +
<img src="https://static.igem.org/mediawiki/parts/f/f7/KR-WT.jpeg" width="60%">
 +
</p>
 +
<p style="font-size:16px;">
 +
    Surely, one should select some ROS-sensitive intracellular localizations, such as mitochondria, plasma membrane, or chromatin to increase efficiency of KillerRed-mediated oxidative stress.
 +
    The following two ways have been found to be effective for killing the eukaryotic cells using KillerRed: (1) via an apoptotic pathway using KillerRed targeted to mitochondria, and (2) via membrane lipid oxidation using membrane-localized KillerRed<sup>[3]</sup>.
 +
 
 +
</p>
 +
<p style="font-size:16px;">
 +
    Under consideration, we decided to fuse a SV40 NLS to the KillerRed protein(<a href="http://parts.igem.org/Part:BBa_K2040122">BBa_K2040122</a>) so that KillerRed can function in the ROS-sensitive intracellular localizations, the chromatin in nucleus, due to the NLS.
 +
</p>
 +
 
 +
<h2 style="margin-top:30px; margin-bottom:10px;">Reference</h2><hr>
 +
 
 +
<p>1. <a hred="https://www.thermofisher.com/tw/zt/home/life-science/cell-culture/transfection/selection/hygromycin-b.html">Hygromycin B | Thermo Fisher Scientific</a></p>  
 +
<p>2. <a hred="http://www.invivogen.com/PDF/Phleomycin_TDS.pdf">Victor Ilyin - Voprosy filosofii i psikhologii - 2015</a></p>
 +
<p>3. <a hred="http://evrogen.com/products/KillerRed/KillerRed_Detailed_description.shtml">Evrogen KillerRed: Detailed description</a></p>
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Latest revision as of 02:00, 20 October 2016

Selection markers

Firstly we tested for effective fungal selection marker hph and ble (Corresponding antibiotics: hygromycin and phleomycin)

The concentration for hygromycin selection ranges from 200-1000 µg/ml[1] and phleomycin-ranges from 10-50 µg/ml [2] for fungi. We chose this series of antibiotic concentration:

Hygromycin (μg/mL): 0, 50, 100, 150, 200

Phleomycin (μg/mL): 0, 25, 50

M. anisopliae were incubated in each of the antibiotics test plates and incubated at 25°C.

Fig.1 Hygromycin test plates (1-5: 0, 50, 100, 150, 200 ug/mL)

Fig.2 Phleomycin test plates (1-3: 0, 25, 50 ug/mL)

As the images shown above, the growing situation of wild type strain M. anisopliae on hygromycin or phleomycin plates are nearly the same comparing to the control. It indicated that our chassis fungi Metarhizium anisopliae ARSEF549 is not sensitive to hygromycin and phleomycin, that means hygromycin and phleomycin can not select transformants during transformation.

Insect hemolymph bioassays

We extracted insect hemolymph from three different species: oriental fruit flies, cherry cockroaches and silkworms.

Fig.3 Oriental fruit flies

Fig.4 Extracting cherry cockroach's hemolymph

Fig.5 Silkworms

After 24 hours(in cherry cockroach and silkworm's hemolymph) and 30 hours(in oriental fruit fly's hemolymph) cultivation, the fungal cells were observed using the bright field microscopy(Magnification: 1000X).

Fig.6 M. anisopliae in oriental fruit fly's hemolymph for 30 hours

Fig.7 M. anisopliae in cherry coach's hemolymph for 24 hours

Fig.8 M. anisopliae in silkworm's hemolymph for 24 hours

Appressorium development can be clearly observed after 24h induction within the hemolymph of silkworms.

Mcl1 promoter

We designed the primers PMcl1-f(ACGTC//CTGCAG//AATCATGCAGCGCTATGAG, with a PstI site underlined) and PMcl1-r(ATAA//GCGGCCGC//CATGATGGTCTAGGGAACG with a NotI site underlined), according to the PMcl1 sequence[1], to amplify the Mcl1 promoter region with Mcl1 mRNA 5'-untranslated region at the 5' end of the coding region. The whole length is 2772bp.

The gel image below shows that we succeed extracting the Mcl1 promoter and its 5'-untranslated region (99bp downstream the promoter) from the genomic DNA of our chassis organism M. anisopliae ARSEF549.

Fig.9 Amplify PMcl1 from gDNA

Then we digested the DNA fragment with NotI and PstI in order to insert it into the backbone. However, when we ran gel electrophoresis to check the digestion result, we found that there is still one unknown PstI cut site inside the PMcl1 region.

Fig.10 The broken PMcl1 fragment

We decided to sequence this DNA fragment we extracted and mutate the PstI site, but we didn't have enough time to finish our relative vectors construction.

KillerRed expression in M. anisopliae

We constructed a KillerRed expression cassette with a fungal promoter PgpdA and a fungal terminator TtrpC. This cassette was used to confirm that KillerRed can be expressed in M. anisopliae

*The fluorescence images below indicated that KillerRed protein was successfully expressed in M. anisopliae.

As we observed, the growth situations of M. anisopliae KR transformants on media will not be affected greatly since irradiation of KillerRed localized in cell cytosol has a weak effect on cell survival in eukaryotic cells[3].

Surely, one should select some ROS-sensitive intracellular localizations, such as mitochondria, plasma membrane, or chromatin to increase efficiency of KillerRed-mediated oxidative stress. The following two ways have been found to be effective for killing the eukaryotic cells using KillerRed: (1) via an apoptotic pathway using KillerRed targeted to mitochondria, and (2) via membrane lipid oxidation using membrane-localized KillerRed[3].

Under consideration, we decided to fuse a SV40 NLS to the KillerRed protein(BBa_K2040122) so that KillerRed can function in the ROS-sensitive intracellular localizations, the chromatin in nucleus, due to the NLS.

Reference

1. Hygromycin B | Thermo Fisher Scientific

2. Victor Ilyin - Voprosy filosofii i psikhologii - 2015

3. Evrogen KillerRed: Detailed description