Difference between revisions of "Team:UChicago/Description"

 
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{{UChicago/Header}}
 
{{UChicago/Header}}
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ul
Line 903: Line 76:
 
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</head>
 
</head>
  
<body bgcolor=yellow lang=EN-US style='tab-interval:.5in'>
+
<body bgcolor=white lang=EN-US link="#0563C1" vlink="#954F72">
  
 
<div class=WordSection1>
 
<div class=WordSection1>
  
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Yay, we can’t believe
+
<p class=MsoNormal><b><span style='font-size:16.0pt;line-height:107%;
you fell for the clickbait. Here’s a picture of a cute cat:<o:p></o:p></b></p>
+
font-family:"Garamond",serif'>Yay, we can’t believe you fell for the clickbait.
 +
Here’s a picture of cute cats:</span></b></p>
  
<p class=MsoNormal><span style='mso-no-proof:yes'><!--[if gte vml 1]><v:shapetype
+
<p class=MsoNormal><b><img width=391 height=503
id="_x0000_t75" coordsize="21600,21600" o:spt="75" o:preferrelative="t"
+
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<v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
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<o:lock v:ext="edit" aspectratio="t"/>
+
</v:shapetype><v:shape id="Picture_x0020_1" o:spid="_x0000_i1026" type="#_x0000_t75"
+
alt="http://www.wallspick.com/wp-content/uploads/2014/12/Grey-Eyes-Cute-Cat.jpg"
+
style='width:187.5pt;height:117pt;visibility:visible;mso-wrap-style:square'>
+
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+
</v:shape><![endif]--><![if !vml]><img width=250 height=156
+
 
src="..//wiki/images/c/c6/UChicago2015Background_filesimage001.jpg"
 
src="..//wiki/images/c/c6/UChicago2015Background_filesimage001.jpg"
alt="http://www.wallspick.com/wp-content/uploads/2014/12/Grey-Eyes-Cute-Cat.jpg"
+
alt="Macintosh HD:Users:benjaminhsu:Library:Containers:com.apple.mail:Data:Library:Mail Downloads:D348DCA2-0D3B-4AAA-B736-06DAD9C3B850:IMG_0834.JPG"></b></p>
v:shapes="Picture_x0020_1"><![endif]></span><b style='mso-bidi-font-weight:
+
normal'><o:p></o:p></b></p>
+
  
<div style='mso-element:para-border-div;border:none;border-bottom:solid windowtext 1.0pt;
+
<div style='border:none;border-bottom:solid windowtext 1.0pt;padding:0in 0in 1.0pt 0in'>
mso-border-bottom-alt:solid windowtext .75pt;padding:0in 0in 1.0pt 0in'>
+
  
<p class=MsoNormal style='border:none;mso-border-bottom-alt:solid windowtext .75pt;
+
<p class=MsoNormal style='border:none;padding:0in'><b><span style='font-size:
padding:0in;mso-padding-alt:0in 0in 1.0pt 0in'><b style='mso-bidi-font-weight:
+
16.0pt;line-height:107%;font-family:"Garamond",serif'> “Are they really going
normal'>Anyways, onto our project:<o:p></o:p></b></p>
+
to cure sleep disorders?! How are they going to do this!?” /
 +
“sigh*……..ponders*”</span></b></p>
  
<p class=MsoNormal style='border:none;mso-border-bottom-alt:solid windowtext .75pt;
+
<p class=MsoNormal style='border:none;padding:0in'><b><span style='font-size:
padding:0in;mso-padding-alt:0in 0in 1.0pt 0in'><b style='mso-bidi-font-weight:
+
16.0pt;line-height:107%;font-family:"Garamond",serif'>&nbsp;</span></b></p>
normal'><o:p>&nbsp;</o:p></b></p>
+
 
 +
<p class=MsoNormal style='border:none;padding:0in'><b><span style='font-size:
 +
16.0pt;line-height:107%;font-family:"Garamond",serif'>Anyways, onto our
 +
project:</span></b></p>
 +
 
 +
<p class=MsoNormal style='border:none;padding:0in'><b>&nbsp;</b></p>
  
 
</div>
 
</div>
  
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><o:p>&nbsp;</o:p></b></p>
+
<p class=MsoNormal><b>&nbsp;</b></p>
  
<p class=MsoNormal align=center style='text-align:center'><b style='mso-bidi-font-weight:
+
<p class=MsoNormal align=center style='text-align:center'><b><span
normal'><span style='font-size:26.0pt;line-height:107%;font-family:"Calibri",sans-serif;
+
style='font-size:26.0pt;line-height:107%;font-family:"Calibri",sans-serif;
mso-ascii-theme-font:minor-latin;mso-hansi-theme-font:minor-latin;color:#C00000'>Background<o:p></o:p></span></b></p>
+
color:#C00000'>Background</span></b></p>
  
<p class=MsoNormal align=center style='text-align:center'><span
+
<p class=MsoNormal align=center style='text-align:center'><img width=625
style='mso-no-proof:yes'><!--[if gte vml 1]><v:shape id="Picture_x0020_2"
+
height=527 id="Picture 2" src="..//wiki/images/5/54/UChicago2015Background_filesimage002.png"></p>
o:spid="_x0000_i1025" type="#_x0000_t75" style='width:468.75pt;height:395.25pt;
+
visibility:visible;mso-wrap-style:square'>
+
<v:imagedata src="..//wiki/images/5/54/UChicago2015Background_filesimage002.png" o:title=""/>
+
</v:shape><![endif]--><![if !vml]><img width=625 height=527
+
src="..//wiki/images/5/54/UChicago2015Background_filesimage002.png" v:shapes="Picture_x0020_2"><![endif]></span><b
+
style='mso-bidi-font-weight:normal'><o:p></o:p></b></p>
+
  
<p class=MsoNormal><i style='mso-bidi-font-style:normal'>Figure 2: Schematic of
+
<p class=MsoNormal align=center style='text-align:center'><i><span
how <span class=SpellE>KaiB</span> and <span class=SpellE>KaiA</span> stimulate
+
style='font-family:"Arial",sans-serif'>Figure 2: Schematic of how KaiB and KaiA
different phosphorylation states of <span class=SpellE>KaiC</span> in a
+
stimulate different phosphorylation states of KaiC in a circadian rhythm. CATS.</span></i></p>
circadian rhythm. CATS.</i><b style='mso-bidi-font-weight:normal'><o:p></o:p></b></p>
+
  
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><o:p>&nbsp;</o:p></b></p>
+
<p class=MsoNormal><b><span style='font-size:16.0pt;line-height:107%;
 +
font-family:"Garamond",serif'>&nbsp;</span></b></p>
  
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>&nbsp;</span></p>
  
<p class=MsoNormal>Circadian rhythms are found in a diverse range of species,
+
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
using a series of oscillatory proteins to govern everything from sleep in humans
+
"Garamond",serif'>Circadian rhythms are found in a diverse range of species,
to cell cycle progression in fungi. The well characterized Kai ABC circadian
+
using a series of oscillatory proteins to govern everything from sleep in
oscillator in cyanobacteria has been demonstrated to regulate photosynthesis,
+
humans to cell cycle progression in fungi. The well characterized Kai ABC
cell division, nitrogen fixation, and a host of other essential cell processes.
+
circadian oscillator in cyanobacteria has been demonstrated to regulate
Because of this wide range of downstream functionality, the ability to
+
photosynthesis, cell division, nitrogen fixation, and a host of other essential
reconstitute this robust oscillator would allow us to control the production of
+
cell processes. Because of this wide range of downstream functionality, the
a target molecule on a 24 hour time scale, such as caffeine or melatonin.</p>
+
ability to reconstitute this robust oscillator would allow us to control the
 +
production of a target molecule on a 24 hour time scale, such as caffeine or
 +
melatonin.</span></p>
  
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>&nbsp;</span></p>
  
<p class=MsoNormal>The Kai ABC system is composed of three proteins, Kai A, Kai
+
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
B, and Kai C. Kai A acts to <span class=SpellE>autophosphorylate</span> Kai C
+
"Garamond",serif'>The Kai ABC system is composed of three proteins, Kai A, Kai
at one of 2 sites, either T431 (T) or S432 (S). Kai B then acts to
+
B, and Kai C. Kai A acts to autophosphorylate Kai C at one of 2 sites, either
auto-dephosphorylate Kai C. Previous research (cite that one paper)has shown
+
T431 (T) or S432 (S). Kai B then acts to auto-dephosphorylate Kai C. Previous
that Kai B and Kai C are present in equal <span class=SpellE>stoichiometries</span>,
+
research (cite that one paper)has shown that Kai B and Kai C are present in
and that the oscillatory character of the system is due to a change in Kai A
+
equal stoichiometries, and that the oscillatory character of the system is due
concentration. </p>
+
to a change in Kai A concentration. </span></p>
  
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>&nbsp;</span></p>
  
<p class=MsoNormal>The 2015 <span class=SpellE>iGem</span> team has <b
+
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
style='mso-bidi-font-weight:normal'>3 main goals</b>:</p>
+
"Garamond",serif'>The 2015 iGem team has <b>3 main goals</b>:</span></p>
  
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1'><![if !supportLists]><span
+
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in'><span
style='font-size:10.0pt;mso-bidi-font-size:12.0pt;line-height:107%;mso-fareast-font-family:
+
style='font-size:10.0pt;line-height:107%;font-family:"Garamond",serif;
"Times New Roman";color:black'><span style='mso-list:Ignore'>1)<span
+
color:black'>1)<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
+
</span></span><span style='font-size:16.0pt;line-height:107%;font-family:"Garamond",serif'> The
style='mso-spacerun:yes'> </span>The first is to reconstitute the Kai ABC
+
first is to reconstitute the Kai ABC oscillator in E. Coli. The oscillator is
oscillator in E. <span class=GramE>Coli</span>. The oscillator is native to
+
native to cyanobacteria, and reconstituting it in the most widely used model
cyanobacteria, and reconstituting it in the most widely used model organism in
+
organism in biomedical research serves as a proof of concept for further
biomedical research serves as a proof of concept for further applications of
+
applications of the oscillatory system. </span></p>
the oscillatory system. </p>
+
  
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l0 level1 lfo1'><![if !supportLists]><span
+
<p class=MsoNormal align=center style='margin-left:.5in;text-align:center'><span
style='font-size:10.0pt;mso-bidi-font-size:12.0pt;line-height:107%;mso-fareast-font-family:
+
style='font-size:16.0pt;line-height:107%;font-family:"Garamond",serif'><img
"Times New Roman";color:black'><span style='mso-list:Ignore'>2)<span
+
width=749 height=336 src="..//wiki/images/9/9c/UChicago2015Background_filesimage003.png"></span></p>
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </span></span></span><![endif]>3)
+
The second goal is to develop a readout system to allow us to assay the
+
downstream effect of the system at various time points during its oscillation
+
by investigating the proteins <span class=SpellE>RpaA</span>, <span
+
class=SpellE>SasA</span>, and <span class=SpellE>CikA</span>. <span
+
class=SpellE>RpaA</span>, <span class=SpellE>SasA</span>, and <span
+
class=SpellE>CikA</span> are components of an expression system directly
+
downstream of <span class=SpellE>KaiABC</span>. <span class=SpellE>RpaA</span>
+
is a master transcription factor that connects the circadian pattern occurring
+
on the protein-protein level in <span class=SpellE>KaiABC</span> to gene
+
expression. <span class=SpellE>SasA</span> and <span class=SpellE>CikA</span>
+
act in similar manner to <span class=SpellE>KaiA</span> and <span class=SpellE>KaiB</span>,
+
<span class=SpellE>phoshporylating</span> and dephosphorylating their substrate
+
<span class=SpellE>RpaA</span> respectively. <span class=SpellE>SasA</span> and
+
<span class=SpellE>CikA</span> directly bind and interact with the <span
+
class=SpellE>KaiABC</span> proteins as well as act on <span class=SpellE>RpaA</span>.
+
While previous studies of transplanting the <span class=SpellE>KaiABC</span>
+
system have demonstrated the ability of <span class=SpellE>SasA</span> and <span
+
class=SpellE>KaiC</span>-P interaction to drive expression, we hope to show
+
oscillations of the <span class=SpellE>RpaA</span> master regulator itself.
+
With the inclusion of the negative regulator <span class=SpellE>CikA</span>, we
+
hope to generate oscillations of <span class=SpellE>RpaA</span> and a
+
downstream <span class=SpellE>kaibc</span> promoter which were not reported in
+
previous studies. </p>
+
  
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in'><span
 +
style='font-size:10.0pt;line-height:107%;font-family:"Garamond",serif;
 +
color:black'>2)<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
</span></span><span style='font-size:16.0pt;line-height:107%;font-family:"Garamond",serif'>The
 +
second goal is to develop a readout system to allow us to assay the downstream
 +
effect of the system at various time points during its oscillation by
 +
investigating the proteins RpaA, SasA, and CikA. RpaA, SasA, and CikA are
 +
components of an expression system directly downstream of KaiABC. RpaA is a
 +
master transcription factor that connects the circadian pattern occurring on
 +
the protein-protein level in KaiABC to gene expression. SasA and CikA act in
 +
similar manner to KaiA and KaiB, phoshporylating and dephosphorylating their
 +
substrate RpaA respectively. SasA and CikA directly bind and interact with the
 +
KaiABC proteins as well as act on RpaA. While previous studies of transplanting
 +
the KaiABC system have demonstrated the ability of SasA and KaiC-P interaction
 +
to drive expression, we hope to show oscillations of the RpaA master regulator
 +
itself. With the inclusion of the negative regulator CikA, we hope to generate
 +
oscillations of RpaA and a downstream kaibc promoter which were not reported in
 +
previous studies. </span></p>
  
<p class=MsoNormal>The third goal is to link the oscillation to the expression
+
<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in'><span
and secretion of AANAT <a style='mso-comment-reference:KY_1;mso-comment-date:
+
style='font-size:10.0pt;line-height:107%;font-family:"Garamond",serif;
20150917T0012'><b style='mso-bidi-font-weight:normal'>(and if ben could explain
+
color:black'>3)<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
more here that would also be great, cause I really have no idea what’s
+
</span></span><span style='font-size:16.0pt;line-height:107%;font-family:"Garamond",serif'>The
happening with this </b></a><span class=GramE><span style='mso-comment-continuation:
+
third goal is to link the oscillation to the expression and secretion of AANAT,
1'><b style='mso-bidi-font-weight:normal'>part )</b></span></span><![if !supportAnnotations]><a
+
an enzyme that catalyzes production of N-Acetylserotonin from tryptophan, with
class=msocomanchor id="_anchor_1"
+
an eventual goal of being able to produce melatonin from tryptophan.</span></p>
onmouseover="msoCommentShow('_anchor_1','_com_1')"
+
onmouseout="msoCommentHide('_com_1')" href="#_msocom_1" language=JavaScript
+
name="_msoanchor_1">[KY1]</a><![endif]><span style='mso-special-character:comment'>&nbsp;</span></p>
+
  
 +
<p class=MsoNormal><b><i><span style='font-size:16.0pt;line-height:107%;
 +
font-family:"Garamond",serif;color:#ED7D31'>&nbsp;</span></i></b></p>
  
<p class=MsoNormal><o:p>&nbsp;</o:p></p>
+
<p class=MsoNormal align=center style='text-align:center'><b><span
 +
style='font-size:16.0pt;line-height:107%;font-family:"Comic Sans MS";
 +
color:black'>Melatonin Biosynthesis Pathway</span></b></p>
  
</div>
+
<p class=MsoNormal><b><i><span style='font-size:16.0pt;line-height:107%;
 +
font-family:"Garamond",serif;color:yellow'>&nbsp;</span></i></b></p>
 +
 
 +
<p class=MsoNormal align=center style='text-align:center'><span
 +
style='font-size:16.0pt;line-height:107%;font-family:"Garamond",serif'><img
 +
width=658 height=929 id="Picture 1" src="..//wiki/images/d/d2/UChicago2015Background_filesimage004.png"
 +
alt="Macintosh HD:Users:benjaminhsu:Desktop:Screen Shot 2015-09-18 at 9.57.14 PM.png"></span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>&nbsp;</span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>&nbsp;</span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>&nbsp;</span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'> </span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>There were a couple reasons why we choose to test melatonin
 +
production control, and in turn AANAT production as our proof-of-concept
 +
project. When we were thinking of things that could be controlled with an
 +
oscillatory system, we had first though of insulin production. It seemed simple
 +
enough, we know that insulin can be produced with bacteria and people with
 +
diabetes need insulin on a timely basis, and now we have a timed delivery
 +
system to possibly deliver it in vivo. However insulin production turned out to
 +
be beyond the scope of our abilities, so we turned to sleep disorders instead,
 +
and since the sleep cycle is mainly controlled by melatonin production, we
 +
decided to focus on this. We had also considered caffeine well, however AANAT
 +
has a very low effective dosage, therefore it is more suitable for our
 +
purposes. </span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'> The biosynthesis of melatonin involves 4 steps, starting
 +
from Tryptophan, and we focused on catalyzing serotonin to N-Acetylserotonin.
 +
Not only is this step the rate-determining step, it is also how our body
 +
naturally controls melatonin production. More importantly, in the context of a
 +
gut-bacteria based drug synthesis system, N-Acetylserotonin is the first
 +
intermediate product that can pass through the blood-brain barrier, also there
 +
would be no shortage of “building blocks”, as most of the serotonin in our body
 +
is in the gut area. </span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>Although we are focusing on the application of AANAT in sleep
 +
medicine, AANAT is also an important part of cancer research as well.</span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>AANAT:</span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>There were a couple reasons why we choose to test melatonin
 +
production control, and in turn AANAT production as our proof-of-concept
 +
project. When we were thinking of things that could be controlled with an
 +
oscillatory system, we had first though of insulin production. It seemed simple
 +
enough, we know that insulin can be produced with bacteria and people with
 +
diabetes need insulin on a timely basis, and now we have a timed delivery
 +
system to possibly deliver it in vivo. However insulin production turned out to
 +
be beyond the scope of our abilities, so we turned to sleep disorders instead,
 +
and since the sleep cycle is mainly controlled by melatonin production, we
 +
decided to focus on this. We had also considered caffeine well, however AANAT
 +
has a very low effective dosage, therefore it is more suitable for our
 +
purposes. </span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'> The biosynthesis of melatonin involves 4 steps, starting
 +
from Tryptophan, and we focused on catalyzing serotonin to N-Acetylserotonin.
 +
Not only is this step the rate-determining step, it is also how our body
 +
naturally controls melatonin production. More importantly, in the context of a
 +
gut-bacteria based drug synthesis system, N-Acetylserotonin is the first
 +
intermediate product that can pass through the blood-brain barrier, also there
 +
would be no shortage of “building blocks”, as most of the serotonin in our body
 +
is in the gut area. <br>
 +
<br>
 +
</span></p>
 +
 
 +
<p class=MsoNormal><span style='font-size:16.0pt;line-height:107%;font-family:
 +
"Garamond",serif'>One thing that is uncertain is whether the AANAT could be
 +
secreted with endogenous secretion systems, and if they can, whether it could
 +
retain its enzyme activity. To test this out we had designed 3 constructs,
 +
unfortunately we did not have time to test them out. To learn more about them,
 +
check out our “what’s next” section<span style='color:black'> in</span><span
 +
style='color:#ED7D31'> </span></span><a
 +
href="https://2015.igem.org/Team:UChicago/Results"><span style='font-size:16.0pt;
 +
line-height:107%;font-family:"Garamond",serif'>Results</span></a><span
 +
style='font-size:16.0pt;line-height:107%;font-family:"Garamond",serif'>.</span></p>
 +
 
 +
<p class=MsoNormal><b><i><span style='font-size:16.0pt;line-height:107%;
 +
font-family:"Garamond",serif;color:#ED7D31'>&nbsp;</span></i></b></p>
 +
 
 +
<p class=MsoNormal><b><i><span style='font-size:16.0pt;line-height:107%;
 +
font-family:"Garamond",serif;color:#ED7D31'>&nbsp;</span></i></b></p>
  
<div style='mso-element:comment-list'><![if !supportAnnotations]>
+
<p class=MsoNormal>&nbsp;</p>
  
<hr class=msocomoff align=left size=1 width="33%">
+
<p class=MsoNormal>   <br>
 +
<br>
 +
<br>
 +
</p>
  
<![endif]>
+
<p class=MsoNormal>&nbsp;</p>
  
<div style='mso-element:comment'><![if !supportAnnotations]>
+
<p class=MsoNormal>&nbsp;</p>
  
<div id="_com_1" class=msocomtxt language=JavaScript
+
<p class=MsoNormal>&nbsp;</p>
onmouseover="msoCommentShow('_anchor_1','_com_1')"
+
onmouseout="msoCommentHide('_com_1')"><![endif]><span style='mso-comment-author:
+
"Kevin Yang";mso-comment-providerid:"Windows Live";mso-comment-userid:dd5da70c2fbc3965'><![if !supportAnnotations]><a
+
name="_msocom_1"></a><![endif]></span>
+
  
<p class=MsoCommentText><span class=MsoCommentReference><span style='font-size:
+
<p class=MsoNormal>&nbsp;</p>
8.0pt'><span style='mso-special-character:comment'>&nbsp;<![if !supportAnnotations]><a
+
href="#_msoanchor_1" class=msocomoff>[KY1]</a><![endif]></span></span></span>
+
  
<![if !supportAnnotations]></div>
+
<p class=MsoNormal>&nbsp;</p>
  
<![endif]></div>
+
<p class=MsoNormal>&nbsp;</p>
  
 
</div>
 
</div>

Latest revision as of 03:54, 9 October 2015

Team UChicago banner.jpg

Yay, we can’t believe you fell for the clickbait. Here’s a picture of cute cats:

Macintosh HD:Users:benjaminhsu:Library:Containers:com.apple.mail:Data:Library:Mail Downloads:D348DCA2-0D3B-4AAA-B736-06DAD9C3B850:IMG_0834.JPG

 “Are they really going to cure sleep disorders?! How are they going to do this!?” / “sigh*……..ponders*”

 

Anyways, onto our project:

 

 

Background

Figure 2: Schematic of how KaiB and KaiA stimulate different phosphorylation states of KaiC in a circadian rhythm. CATS.

 

 

Circadian rhythms are found in a diverse range of species, using a series of oscillatory proteins to govern everything from sleep in humans to cell cycle progression in fungi. The well characterized Kai ABC circadian oscillator in cyanobacteria has been demonstrated to regulate photosynthesis, cell division, nitrogen fixation, and a host of other essential cell processes. Because of this wide range of downstream functionality, the ability to reconstitute this robust oscillator would allow us to control the production of a target molecule on a 24 hour time scale, such as caffeine or melatonin.

 

The Kai ABC system is composed of three proteins, Kai A, Kai B, and Kai C. Kai A acts to autophosphorylate Kai C at one of 2 sites, either T431 (T) or S432 (S). Kai B then acts to auto-dephosphorylate Kai C. Previous research (cite that one paper)has shown that Kai B and Kai C are present in equal stoichiometries, and that the oscillatory character of the system is due to a change in Kai A concentration.

 

The 2015 iGem team has 3 main goals:

1)        The first is to reconstitute the Kai ABC oscillator in E. Coli. The oscillator is native to cyanobacteria, and reconstituting it in the most widely used model organism in biomedical research serves as a proof of concept for further applications of the oscillatory system.

2)       The second goal is to develop a readout system to allow us to assay the downstream effect of the system at various time points during its oscillation by investigating the proteins RpaA, SasA, and CikA. RpaA, SasA, and CikA are components of an expression system directly downstream of KaiABC. RpaA is a master transcription factor that connects the circadian pattern occurring on the protein-protein level in KaiABC to gene expression. SasA and CikA act in similar manner to KaiA and KaiB, phoshporylating and dephosphorylating their substrate RpaA respectively. SasA and CikA directly bind and interact with the KaiABC proteins as well as act on RpaA. While previous studies of transplanting the KaiABC system have demonstrated the ability of SasA and KaiC-P interaction to drive expression, we hope to show oscillations of the RpaA master regulator itself. With the inclusion of the negative regulator CikA, we hope to generate oscillations of RpaA and a downstream kaibc promoter which were not reported in previous studies.

3)       The third goal is to link the oscillation to the expression and secretion of AANAT, an enzyme that catalyzes production of N-Acetylserotonin from tryptophan, with an eventual goal of being able to produce melatonin from tryptophan.

 

Melatonin Biosynthesis Pathway

 

Macintosh HD:Users:benjaminhsu:Desktop:Screen Shot 2015-09-18 at 9.57.14 PM.png

 

 

 

 

There were a couple reasons why we choose to test melatonin production control, and in turn AANAT production as our proof-of-concept project. When we were thinking of things that could be controlled with an oscillatory system, we had first though of insulin production. It seemed simple enough, we know that insulin can be produced with bacteria and people with diabetes need insulin on a timely basis, and now we have a timed delivery system to possibly deliver it in vivo. However insulin production turned out to be beyond the scope of our abilities, so we turned to sleep disorders instead, and since the sleep cycle is mainly controlled by melatonin production, we decided to focus on this. We had also considered caffeine well, however AANAT has a very low effective dosage, therefore it is more suitable for our purposes.

 The biosynthesis of melatonin involves 4 steps, starting from Tryptophan, and we focused on catalyzing serotonin to N-Acetylserotonin. Not only is this step the rate-determining step, it is also how our body naturally controls melatonin production. More importantly, in the context of a gut-bacteria based drug synthesis system, N-Acetylserotonin is the first intermediate product that can pass through the blood-brain barrier, also there would be no shortage of “building blocks”, as most of the serotonin in our body is in the gut area.

Although we are focusing on the application of AANAT in sleep medicine, AANAT is also an important part of cancer research as well.

AANAT:

There were a couple reasons why we choose to test melatonin production control, and in turn AANAT production as our proof-of-concept project. When we were thinking of things that could be controlled with an oscillatory system, we had first though of insulin production. It seemed simple enough, we know that insulin can be produced with bacteria and people with diabetes need insulin on a timely basis, and now we have a timed delivery system to possibly deliver it in vivo. However insulin production turned out to be beyond the scope of our abilities, so we turned to sleep disorders instead, and since the sleep cycle is mainly controlled by melatonin production, we decided to focus on this. We had also considered caffeine well, however AANAT has a very low effective dosage, therefore it is more suitable for our purposes.

 The biosynthesis of melatonin involves 4 steps, starting from Tryptophan, and we focused on catalyzing serotonin to N-Acetylserotonin. Not only is this step the rate-determining step, it is also how our body naturally controls melatonin production. More importantly, in the context of a gut-bacteria based drug synthesis system, N-Acetylserotonin is the first intermediate product that can pass through the blood-brain barrier, also there would be no shortage of “building blocks”, as most of the serotonin in our body is in the gut area.

One thing that is uncertain is whether the AANAT could be secreted with endogenous secretion systems, and if they can, whether it could retain its enzyme activity. To test this out we had designed 3 constructs, unfortunately we did not have time to test them out. To learn more about them, check out our “what’s next” section in Results.

 

 

 

  


 

 

 

 

 

 



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