Team:NYMU-Taipei/results/immunological-solution1

From 2011.igem.org

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==<font size=5><font color=crimson>'''Constructs & Parts '''</font></font>==
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==<font size=5><font color=crimson>'''Construction & Parts '''</font></font>==
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===<font size=4><font color=green>'''(1) <i>MinC</i> Construct and <i>Inv</i> & <i>LLO</i> Constructs on AMB-1'''</font></font>===
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<center>
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<font size=4><font color=green>'''Our <i>minC</i> Construct and <i>Inv</i> & <i>LLO</i> Construct on AMB-1'''</font></font>
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[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624053 BBa_K624053]
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{| class="wikitable" border="1"
{| class="wikitable" border="1"
|-
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!  [[Image: AMB-1_minC_NYMU.jpg|center]]
!  [[Image: AMB-1_minC_NYMU.jpg|center]]
!  [[Image: AMB-1_Inv_LLO.jpg|center]]
!  [[Image: AMB-1_Inv_LLO.jpg|center]]
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|- style="text-align:center;"
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|  <font size=3>Fig. Design of <i>minC</i> construct on AMB-1<br>Backbone plasmid: pYMB</font>
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|  <font size=3>Fig. Design of <i>Inv</i> & <i>LLO</i> construct on AMB-1<br>Backbone plasmid: pRKm415</font>
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|}
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</center>
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===<font size=4><font color=green>'''(2) Related Parts'''</font></font>===
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<font size=3>We have created several parts on the basis of Biobrick. The expression will be shown on the partregistry webpage.
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{| border=1
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|rowspan=1 |  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624033 '''<u>BBa_K624033</u>''']
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|<i>minC</i> cell division inhibitor (revised)
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|-
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|rowspan=1 |  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624034 '''<u>BBa_K624034</u>''']
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|revised <i>minC</i> primer (forward)
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|-
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|rowspan=1 |  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624035 '''<u>BBa_K624035</u>''']
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|revised <i>minC</i> primer (reverse)
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|-
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| rowspan=1 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624036 '''<u> BBa_K624036</u>''']
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|<i>minC</i>+<i>mcherry</i>
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|-
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| rowspan=1 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624037 '''<u>BBa_K624037</u>''']
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| <i>rbs</i>+<i>minC</i>+<i>mcherry</i>
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|-
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| rowspan=1 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624040 '''<u>BBa_K624040</u>''']
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|<i>pT7-tetO</i>+<i>rbs</i>+<i>minC</i>
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|-
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| rowspan=1 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624045 '''<u>BBa_K624045</u>''']
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|<i>Pmsp1(tetO)</i> + <i>rbs(Pmsp3)</i> + <i>minC</i> + <i>rbs(Pmsp3)</i> + <i>mcherry</i>
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|-
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| rowspan=1 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624056 '''<u>BBa_K624056</u>''']
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| pYMB essentials + <i>rbs(Pmsp3)</i> + <i>minC</i>
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|-
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| rowspan=1 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624057 '''<u>BBa_K624057</u>''']
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| pYMB essentials + <i>rbs(Pmsp3)</i> + <i>LLO</i>
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|-
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| rowspan=1 | [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624058 '''<u>BBa_K624058</u>''']
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| pYMB essentials + <i>rbs(Pmsp3)</i> + <i>Inv</i>
|-
|-
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|  <center>Fig. Design of <i>minC</i> Construct on AMB-1</center><center>Backbone Plasmid: pYMB</center>
 
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|  <center>Fig. Design of <i>Inv</i> & <i>LLO</i> Construct on AMB-1</center><center>Backbone Plasmid: pRKm415</center>
 
|}
|}
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</font>
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==<font size=5><font color=crimson>'''Symbiosis with Human Glial Cell '''</font></font>==
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===<font size=4><font color=green>'''(3) Nested-PCR Primers for Various Strains'''</font></font>===
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<font size=3>It is well-studied that <i>Magnetotactic bacteria be introduced into guanulocytes and monocytes by phagocytosis.</i>(Tadashi Matsunaga et all. 1986) In order to simulate the circumstances in brain, we put <i>E.coli</i> and <i>AMB-1</i> separately into mixed glial cells <i>in vitro</i>. Below are the photos we captured under microscope.</font>
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<font size=4><font color=blue>'''(i) nested-Inv'''</font></font>
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<font size=3>
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{| border=1
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|rowspan=1 |  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624063 '''<u>BBa_K624063</u>''']
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|nested_Inv F (inter-strain nested primer)
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|-
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|rowspan=1 |  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624064 '''<u>BBa_K624064</u>''']
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|nested_Inv R (inter-strain nested primer)
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|}
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</font>
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'''<font size=3><font color=red>check out [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624032 BBa_K624032] for detailed characterization</font></font>'''
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<font size=4><font color=blue>'''(ii) nested-LLO'''</font></font>
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<font size=3>
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{| border=1
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|rowspan=1 |  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624065 '''<u>BBa_K624065</u>''']
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|nested_LLO F (inter-strain nested primer)
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|-
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|rowspan=1 |  [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624066 '''<u>BBa_K624066</u>''']
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|nested_LLO R (inter-strain nested primer)
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|}
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</font>
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{| class="wikitable" border="1"
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===<font size=4><font color=green>'''(4) Results of PCR from genomic DNA'''</font></font>===
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<center>
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{| class="wikitable" border="0"
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!  [[Image: AMB-1_glial1.tif|center]]
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!  [[Image:gel_invasin.png]]
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!  [[Image: AMB-1_glial2.tif|center]]
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!  [[Image:gel_LLO.png]]
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!  [[Image:gel_minC.png]]
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|- style="text-align:center;"
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|  <font size=3>Fig. <i>invasin</i> PCR cloned from<br><i>Yersinia enterocolitica</i></font>
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| <font size=3>Fig. <i>LLO</i> PCR cloned from<br><i>Listeria monocytogenes</i></font>
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|  <font size=3>Fig. <i>minC</i> PCR cloned from<br><i>E.coli</i> K12 MG1655</font>
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|}
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</center>
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===<font size=4><font color=green>'''(5) Expression of TetR Protein Enabled Regulation'''</font></font>===
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<center>
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{| class="wikitable" border="0"
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| [[Image: glial1.tif]]
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! [[Image:expression_of_tetR_protein.png]]
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|  [[Image: glial2.tif]]
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|-
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|  <font size=3>Fig. From the left to right, there are separately <b>marker</b> labelled at 72kDa and 26kDa, <br><b>supernatant control</b>,  <b>supernatant experiment</b>, <b>pellet control</b>, <b>pellet experiment</b>.</font>
|}
|}
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</center>
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<font size=3>Tetracyclin resistent protein and GFP were induced by IPTG to express in BL-21 <i>E.coli</i> strain for the confirmation of the construct.As SDS-PAGE shown above, there is no difference between supernatant control and experiment while tetracyclin resistent protein and GFP express in the pellet experiment.It is proved that the expression of tetracyclin resistent protein can enable the regulation.</font>
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==<font size=5><font color=crimson>'''Measurements of Construct <i>tetR</i>+<i>rbs</i>+<i>gfp</i> under Fluorescence Microscope'''</font></font>==
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==<font size=5><font color=crimson>'''Symbiosis within Human Glial Cells '''</font></font>==
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===<font size=4><font color=green>'''<b>(1)</b> <font size=3>Magnetotactic bacteria were introduced into guanulocytes and monocytes by phagocytosis.(Tadashi Matsunaga et all. 1986)</font></font>===
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<center>
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{| class="wikitable" border="0"
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|-
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!  [[Image:monocyte.jpg]]
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|- style="text-align:center;"
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|  <font size=3>Fig.The transmission electron micrograph(TEM) of a monocyte <br>that had ingested magnetotactic bacteria. The bar represents 5 μm.</font>
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|}
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</center>
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===<font size=4><font color=green>'''<b>(2)</b> In our experiment, AMB-1 was introduced into primary mixed glial cells <i>in vitro</i>. Figures 1~4 show the photos captured under the microscope.</font></font>===
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'''<font size=3>Check out [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624032 BBa_K624032] for detailed characterization</font>'''
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<center>
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{| class="wikitable" border="1"
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|- style="text-align:center;"
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|  [[Image: AMB-1_glial1.jpg|center|400px|thumb|<font size=3>Fig.1 primary mixed glial cell introduced with AMB-1 1</font>]]
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|  [[Image: AMB-1_glial2.jpg|center|400px|thumb|<font size=3>Fig.2 primary mixed glial cell introduced with AMB-1 2</font>]]
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|- style="text-align:center;"
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|  [[Image: glial1.jpg|center|400px|thumb|<font size=3>Fig.3 primary mixed glial cell control 1</font>]]
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|  [[Image: glial2.jpg|center|400px|thumb|<font size=3>Fig.4 primary mixed glial cell control 2</font>]]
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|}
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</center>
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==<font size=5><font color=crimson>'''Measurements of Construct <i>tetR</i>+<i>rbs</i>+<i>gfp</i> under Fluorescence Microscope'''</font></font>==
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<center>
{| class="wikitable" border="0"
{| class="wikitable" border="0"
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</center>
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<font size=3>These are the measurements of our construct [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624001 '''<u><i>tetR</i>+<i>rbs</i>+<i>GFP</i></u>]'''(BBa_K624001) under fluorescence microscope. '''The picture on the left''' is from the control group of ''<i>E. coli</i>'' strain DH5-alpha. '''The middle''' is <i>tetR</i>+<i>rbs</i>+<i>gfp</i> in plasmid pSB1C3 in ''<i>E. coli</i>'', while '''the right''' is from a plate without any bacilli. All graphs are measured in fixed exposure time. Construct of <i>tetR</i>+<i>rbs</i>+<i>gfp</i> which emits light is thought to be a leak phenomenon of plasmid, while RE digest has proved that the sequence length is correct, and further sequencing work is under operation.
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<font size=3>These are the measurements of our construct [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624001 '''<u><i>tetR</i>+<i>rbs</i>+<i>GFP</i></u>]'''(BBa_K624001) under fluorescence microscope. '''The left graph''' is from the control group of ''<i>E. coli</i>'' strain DH5-alpha. '''The middle''' is <i>tetR</i>+<i>rbs</i>+<i>gfp</i> in plasmid pSB1C3 in ''<i>E. coli</i>'', while '''the right''' is from a plate without any bacilli. All graphs are measured in fixed exposure time. Construct of <i>tetR</i>+<i>rbs</i>+<i>gfp</i> which emits light is thought to be a leak phenomenon of plasmid, while RE digest has proved that the sequence length is correct, and further sequencing work is under operation.
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<font size=4><font color=green>'''<i>gfp</i> measurements under different excitation wavelengths'''</font></font>
<font size=4><font color=green>'''<i>gfp</i> measurements under different excitation wavelengths'''</font></font>
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<center>
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[[Image:33.JPG]]
[[Image:33.JPG]]
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</center>
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<font size=3>The expression of <i>gfp</i> is measured precisely under excitation wavelength from 475nm to 501nm, given the absorption wavelength 511nm.  In our measurement, wavelength over 493nm yields high background, resulting in values of control group surging far beyond normal condition.  Thus, <font size=3><font color=mediumblue>'''493nm is suggested as the best excitation wavelength'''</font></font>, as it presents the highest emission light and the lowest of the control group.
<font size=3>The expression of <i>gfp</i> is measured precisely under excitation wavelength from 475nm to 501nm, given the absorption wavelength 511nm.  In our measurement, wavelength over 493nm yields high background, resulting in values of control group surging far beyond normal condition.  Thus, <font size=3><font color=mediumblue>'''493nm is suggested as the best excitation wavelength'''</font></font>, as it presents the highest emission light and the lowest of the control group.

Latest revision as of 21:51, 28 October 2011

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Contents

Constructs & Parts

(1) MinC Construct and Inv & LLO Constructs on AMB-1

AMB-1 minC NYMU.jpg
AMB-1 Inv LLO.jpg
Fig. Design of minC construct on AMB-1
Backbone plasmid: pYMB 		Fig. Design of Inv & LLO construct on AMB-1
Backbone plasmid: pRKm415

(2) Related Parts

We have created several parts on the basis of Biobrick. The expression will be shown on the partregistry webpage.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624033 BBa_K624033] minC cell division inhibitor (revised)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624034 BBa_K624034] revised minC primer (forward)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624035 BBa_K624035] revised minC primer (reverse)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624036 BBa_K624036] minC+mcherry
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624037 BBa_K624037] rbs+minC+mcherry
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624040 BBa_K624040] pT7-tetO+rbs+minC
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624045 BBa_K624045] Pmsp1(tetO) + rbs(Pmsp3) + minC + rbs(Pmsp3) + mcherry
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624056 BBa_K624056] pYMB essentials + rbs(Pmsp3) + minC
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624057 BBa_K624057] pYMB essentials + rbs(Pmsp3) + LLO
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624058 BBa_K624058] pYMB essentials + rbs(Pmsp3) + Inv

(3) Nested-PCR Primers for Various Strains

(i) nested-Inv

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624063 BBa_K624063] nested_Inv F (inter-strain nested primer)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624064 BBa_K624064] nested_Inv R (inter-strain nested primer)

(ii) nested-LLO

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624065 BBa_K624065] nested_LLO F (inter-strain nested primer)
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K624066 BBa_K624066] nested_LLO R (inter-strain nested primer)

(4) Results of PCR from genomic DNA

Gel invasin.png Gel LLO.png Gel minC.png
Fig. invasin PCR cloned from
Yersinia enterocolitica 		Fig. LLO PCR cloned from
Listeria monocytogenes 		Fig. minC PCR cloned from
E.coli K12 MG1655

(5) Expression of TetR Protein Enabled Regulation

Expression of tetR protein.png
Fig. From the left to right, there are separately marker labelled at 72kDa and 26kDa,
supernatant control, supernatant experiment, pellet control, pellet experiment.

Tetracyclin resistent protein and GFP were induced by IPTG to express in BL-21 E.coli strain for the confirmation of the construct.As SDS-PAGE shown above, there is no difference between supernatant control and experiment while tetracyclin resistent protein and GFP express in the pellet experiment.It is proved that the expression of tetracyclin resistent protein can enable the regulation.

Symbiosis within Human Glial Cells

(1) Magnetotactic bacteria were introduced into guanulocytes and monocytes by phagocytosis.(Tadashi Matsunaga et all. 1986)

Monocyte.jpg
Fig.The transmission electron micrograph(TEM) of a monocyte
that had ingested magnetotactic bacteria. The bar represents 5 μm.

(2) In our experiment, AMB-1 was introduced into primary mixed glial cells in vitro. Figures 1~4 show the photos captured under the microscope.

Check out [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624032 BBa_K624032] for detailed characterization

Fig.1 primary mixed glial cell introduced with AMB-1 1
Fig.2 primary mixed glial cell introduced with AMB-1 2
Fig.3 primary mixed glial cell control 1
Fig.4 primary mixed glial cell control 2

Measurements of Construct tetR+rbs+gfp under Fluorescence Microscope

30.JPG 31.JPG 32.JPG

These are the measurements of our construct [http://partsregistry.org/wiki/index.php?title=Part:BBa_K624001 tetR+rbs+GFP](BBa_K624001) under fluorescence microscope. The picture on the left is from the control group of E. coli strain DH5-alpha. The middle is tetR+rbs+gfp in plasmid pSB1C3 in E. coli, while the right is from a plate without any bacilli. All graphs are measured in fixed exposure time. Construct of tetR+rbs+gfp which emits light is thought to be a leak phenomenon of plasmid, while RE digest has proved that the sequence length is correct, and further sequencing work is under operation.

gfp measurements under different excitation wavelengths

33.JPG

The expression of gfp is measured precisely under excitation wavelength from 475nm to 501nm, given the absorption wavelength 511nm. In our measurement, wavelength over 493nm yields high background, resulting in values of control group surging far beyond normal condition. Thus, 493nm is suggested as the best excitation wavelength, as it presents the highest emission light and the lowest of the control group.

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