Team:UCL/Project/Circuit

From 2013.igem.org

(Difference between revisions)
 
(43 intermediate revisions not shown)
Line 48: Line 48:
<div class="full_page">
<div class="full_page">
-
<div class="main_image"></div>
+
<div class="small_image_right" style="background-image:url('http://2013.igem.org/wiki/images/f/f7/1264308_10151755819728591_1663144720_o.jpg');height:680px;width:644px"></div>
<p class="major_title">CIRCUIT OVERVIEW</p>
<p class="major_title">CIRCUIT OVERVIEW</p>
<p class="minor_title">IGEM: Intelligently Genetically Engineered Microglia</p>
<p class="minor_title">IGEM: Intelligently Genetically Engineered Microglia</p>
<p class="body_text">
<p class="body_text">
-
Our <a href="http://2013.igem.org/Team:UCL/Project" target="_blank">ambitious project</a> concerns bringing synthetic biology to the brain in order to try a novel approach to tackling  <a href="http://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s Disease (AD)</a>. <a href="http://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">Microglia</a> are mobile brain cells, making them an ideal <a href="http://2013.igem.org/Team:UCL/Project/Chassis" target="_blank">chassis</a>. To do this, our proposed treatment would involve extracting microglia from a patient, or using a specially bred immortalised line of human microglia, to avoid rejection, and transfecting it with our new genetic circuit. Implantation into the brain could be performed surgically or using a viral vector - but in order to better control the numbers of genetically engineered microglia (GEM) in the brain micro-neurosurgery may prove best. Our circuit is designed to detect <a href="http://2013.igem.org/Team:UCL/Project/Detection" target="_blank">detect</a> <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">amyloid plaques</a>, attract other microglia, <a href="http://2013.igem.org/Team:UCL/Project/Degradation" target="_blank">degrade</a> the plaques, <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">reduce neuroinflammation and support </a> dying neurons. <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">Theoretically</a>, this should halt the progression of Alzheimer’s disease and could lead to other forms of <a href="http://2013.igem.org/Team:UCL/Practice" target="_blank">neuro-genetic engineering</a>.
+
Our <a href="http://2013.igem.org/Team:UCL/Project" target="_blank">ambitious project</a> concerns bringing synthetic biology to the brain in order to try a novel approach to tackling  <a href="http://2013.igem.org/Team:UCL/Background/Alzheimers" target="_blank">Alzheimer’s Disease (AD)</a>. <a href="http://2013.igem.org/Team:UCL/Background/Microglia" target="_blank">Microglia</a> are mobile brain cells, making them an ideal <a href="http://2013.igem.org/Team:UCL/Project/Chassis" target="_blank">chassis</a>. To do this, our proposed treatment would involve extracting microglia from a patient, or using a specially bred immortalised line of human microglia, to avoid rejection, and transfecting it with our new genetic circuit. Alternatively, we could use <a href="http://2013.igem.org/Team:UCL/Project/Chemotaxis" target="_blank">nano-complexes</a> to transfect native microglia. Implantation of genetically engineered microglia(GEM) or DNA into the brain would be performed via micro-neurosurgery. Our circuit is designed to <a href="http://2013.igem.org/Team:UCL/Project/Detection" target="_blank">detect</a> <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">amyloid plaques</a>, attract other microglia, <a href="http://2013.igem.org/Team:UCL/Project/Degradation" target="_blank">degrade</a> the plaques, <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">reduce neuroinflammation and support </a> dying neurons. <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">Theoretically</a>, this should halt the progression of Alzheimer’s disease and could lead to other forms of <a href="http://2013.igem.org/Team:UCL/Practice" target="_blank">neuro-genetic engineering</a>.
</p>
</p>
<p class="body_text">
<p class="body_text">
-
Our GEM have zeocin resistance would be injected into the key areas of pathology in the AD brain; the neocortex, limbic structures, hippocampus, amygdala, and some of the brainstem nuclei. Microglia are naturally drawn towards the senile plaques characteristic of AD. Senile plaques create <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">free radicals</a>, so as the GEM near the plaques oxidative stress increases. In response, our oxidative stress promoter will increase the transcription of key genes. Their protein products are <a href="http://2013.igem.org/Team:UCL/Project/Degradation" target="_blank">matrix metalloproteinase 9 (MMP-9)</a> , <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">vasoactive intestinal peptide(VIP)</a>, <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">brain derived neurotrophic factor (BDNF)</a> and  a chemoattractant called <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">interferon gamma-induced protein 10(IP-10)</a>.
+
Our GEM, selected for using zeocin resistance, would be injected into the key areas of pathology in the AD brain; the neocortex, limbic structures, hippocampus, amygdala, and some of the brainstem nuclei. Microglia are naturally drawn towards the senile plaques characteristic of AD. Senile plaques create <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">free radicals</a>, so as the GEM near the plaques oxidative stress increases. In response, our oxidative stress promoter will increase the transcription of key genes. Their protein products are <a href="http://2013.igem.org/Team:UCL/Project/Degradation" target="_blank">matrix metalloproteinase 9 (MMP-9)</a> , <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">vasoactive intestinal peptide(VIP)</a>, <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">brain derived neurotrophic factor (BDNF)</a> and  a chemoattractant called <a href="http://2013.igem.org/Team:UCL/Project/Developments" target="_blank">interferon gamma-induced protein 10(IP-10)</a>.
</p>
</p>
 +
<p class="body_text">
<p class="body_text">
Brain cell death in AD is engendered by <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">neuroinflammation</a> due to microglia activation. VIP is able to de-activate GEM and native microglia. Activated microglia do not, however, produce proteases to clear plaques or attract other microglia. MMP-9 is capable of degrading β-amyloid fibrils and soluble β-amyloid in situ <a href="http://www.ncbi.nlm.nih.gov/pubmed/16787929" target="_blank">(Yan et al. 2006)</a>. <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">Many theories</a> of AD causation see amyloid build up as key to pathogenesis. Increasing clearance could therefore halt disease progression, and synthetically expressing MMP-9 in volume will ensure greater than natural plaque degradation even after microglial inactivation. In order attract more GEM to increase clearance rate, the chemoattractant is produced. AD may also arise due to insiufficient BDNF signaling <a href="http://www.ncbi.nlm.nih.gov/pubmed/20436277" target="_blank">(Frade & Lopez-Sanchez 2010)</a>, and BDNF is able to support dying neurons, which is why we also propose its production.  
Brain cell death in AD is engendered by <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">neuroinflammation</a> due to microglia activation. VIP is able to de-activate GEM and native microglia. Activated microglia do not, however, produce proteases to clear plaques or attract other microglia. MMP-9 is capable of degrading β-amyloid fibrils and soluble β-amyloid in situ <a href="http://www.ncbi.nlm.nih.gov/pubmed/16787929" target="_blank">(Yan et al. 2006)</a>. <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">Many theories</a> of AD causation see amyloid build up as key to pathogenesis. Increasing clearance could therefore halt disease progression, and synthetically expressing MMP-9 in volume will ensure greater than natural plaque degradation even after microglial inactivation. In order attract more GEM to increase clearance rate, the chemoattractant is produced. AD may also arise due to insiufficient BDNF signaling <a href="http://www.ncbi.nlm.nih.gov/pubmed/20436277" target="_blank">(Frade & Lopez-Sanchez 2010)</a>, and BDNF is able to support dying neurons, which is why we also propose its production.  
Line 62: Line 63:
</div>
</div>
 +
<div class="gap"></div>
<div class="gap"></div>
Line 68: Line 70:
<div class="row_small">
<div class="row_small">
-
<div class="part"></div>
+
<div class="part" style="background-image:url('http://2013.igem.org/wiki/images/4/4c/Screen_Shot_2013-09-27_at_16.38.49.png');"></div>
<div class="description">
<div class="description">
<p class="body_text"><b>
<p class="body_text"><b>
-
Zeocin is a glycopeptide antibiotic capable of killing most bacteria, fungi, yeast, plant, and animal cells by intercalating DNA and inducing double strand breakage. This makes zeocin resistance an ideal selective maker for our project, which involves both bacterial and mammalian chassis. The product of the Sh ble gene, isolated from the bacterium Streptoalloteichus hindustanus <a href="http://www.ncbi.nlm.nih.gov/pubmed/2450783" target="_blank">(Gatignol et al. 1988)</a>, confers zeocin resistance to transfected/transformed cells. Sh ble is a small binding protein with strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA.
+
Zeocin is a glycopeptide antibiotic capable of killing most bacteria, fungi, yeast, plant, and animal cells by intercalating DNA and inducing double strand breakage. This makes zeocin resistance an ideal selective maker for our project, which involves both bacterial and mammalian chassis. The product of the ''Sh ble'' gene, isolated from the bacterium Streptoalloteichus hindustanus <a href="http://www.ncbi.nlm.nih.gov/pubmed/2450783" target="_blank">(Gatignol et al. 1988)</a>, confers zeocin resistance to transfected/transformed cells. Sh ble is a small binding protein with strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA.
</p>
</p>
</div>
</div>
Line 80: Line 82:
<p class="major_title">Oxidative Stress Promoter</p>
<p class="major_title">Oxidative Stress Promoter</p>
<div class="row_small">
<div class="row_small">
-
<div class="part"></div>
+
<div class="part" style="background-image:url('http://2013.igem.org/wiki/images/2/24/NFKB_png2.png');"></div>
<div class="description">
<div class="description">
<p class="body_text"><b>
<p class="body_text"><b>
-
Oxidative stress via free radical production increases with proximity to senile plaques <a href="http://www.ncbi.nlm.nih.gov/pubmed/10863548" target="_blank">(Colton et al., 2000)</a>. The microglia immune response around plaques also increases oxidative stress. Therefore, we have designed a promoter which will initiate transcription in response to oxidative stress to ensure the production of key proteins only in the plaques’ locales. This promoter is an improvement of a yeast minimal promoter (<a href="http://parts.igem.org/Part:BBa_K105027" target="_blank">cyc100</a>) already in the registry. NF-κB is a transcription factor which translocates to the nucleus under oxidative stress <a href="http://www.ncbi.nlm.nih.gov/pubmed/12730877" target="_blank">(Shi et al., 2003)</a>, and binds to the sequence GGGAATTT <a href="http://www.ncbi.nlm.nih.gov/pubmed/19435890" target="_blank">(Park et al., 2009)</a>. Thus, by placing this site upstream of the yeast minimal promoter, we created a novel mammalian promoter which initiates transcription in response to oxidative stress.
+
Oxidative stress via free radical production increases with proximity to senile plaques <a href="http://www.ncbi.nlm.nih.gov/pubmed/10863548" target="_blank">(Colton et al., 2000)</a>. The microglia immune response around plaques also increases oxidative stress. Therefore, we have designed a promoter which will initiate transcription in response to oxidative stress to ensure the production of key proteins only in the plaques’ locales. This promoter is an improvement of a yeast minimal promoter (<a href="http://parts.igem.org/Part:BBa_K105027" target="_blank">cyc100</a>) already in the registry. NF-κB is a transcription factor (pictured to the left) which translocates to the nucleus under oxidative stress <a href="http://www.ncbi.nlm.nih.gov/pubmed/12730877" target="_blank">(Shi et al., 2003)</a>, and binds to the sequence GGGAATTT <a href="http://www.ncbi.nlm.nih.gov/pubmed/19435890" target="_blank">(Park et al., 2009)</a>. Thus, by placing this site upstream of the yeast minimal promoter, we created a novel mammalian promoter which initiates transcription in response to oxidative stress.
</div>
</div>
</div>
</div>
 +
 +
<div class="gap"></div>
 +
<p class="major_title">Active MMP-9</p>
 +
<div class="row_small">
 +
<div class="part" style="background-image:url('http://2013.igem.org/wiki/images/1/1b/MMP9_png2.png');"></div>
 +
<div class="description">
 +
<p class="body_text"><b>
 +
MMP-9, also known as gelatinase B, is most commonly known for its role in breaking down the extracellular matrix. Naturally it is secreted in its inactive form and must be cleaved by other proteases, but our GEM are are meant to produce just the active form. It has been shown by Yan et al. that MMP-9 is the only known endogenous protease that degrades both fibrillar and soluble forms of amyloid-β peptide (Aβ). This satisfies the <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">'Amyloid Cascade Hypothesis'</a> as well as theories that see plaques as <a href="http://2013.igem.org/Team:UCL/Background/Neuropathology" target="_blank">neuroprotective</a>, and soluble Aβ as the real threat. MMP-9 is expressed at low basal levels in microglia and may keep plaque size in dynamic equilibrium <a href="http://www.ncbi.nlm.nih.gov/pubmed/16787929" target="_blank">(Yan et al. 2006)</a>. Over producing it in (inactive) GEM could greatly improve both soluble and insoluble Aβ clearance. MMP-9 must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could cause damage to brain tissue if, for example, injected into the brain.
 +
</div>
 +
</div>
 +
 +
 +
<div class="gap"></div>
 +
<p class="major_title">VIP</p>
 +
<div class="row_small">
 +
<div class="part" style="background-image:url('http://2013.igem.org/wiki/images/9/93/VIP_png2.png');"></div>
 +
<div class="description">
 +
<p class="body_text"><b>
 +
This is a hormone containing 28 amino acid residues. It is a powerful anti-inflammatory neuropeptide. It exhibits neuroprotection in AD model systems <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC40251/" target="_blank">(Goze et al. 1996)</a>, able to save neurons exposed to excessive defective Aβ (70% success rate <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC40251/" target="_blank">(Goze et al. 1996)</a> by limiting neuroinflammation). VIP de-activates microglia <a href="http://www.ncbi.nlm.nih.gov/pubmed/12923064" target="_blank">(Delgado and Ganea 2003)</a>, inhibiting the production of inflammatory chemokines from microglia and reduces chemotaxis. VIP must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could inactivate too much of the brain immune if, for example, injected into the brain. If constitutively expressed, it would stop GEM migrating to plaques. 
 +
</div>
 +
</div>
 +
 +
<div class="gap"></div>
 +
<p class="major_title">BDNF</p>
 +
<div class="row_small">
 +
<div class="part" style="background-image:url('http://2013.igem.org/wiki/images/5/56/BDNF_pndfsdfg2.png');"></div>
 +
<div class="description">
 +
<p class="body_text"><b>
 +
While there is no change in BDNF levels in AD patients <a href="http://www.ncbi.nlm.nih.gov/pubmed/19363274" target="_blank">(O’Bryant et al. 2009)</a>, oxidative stress in older brains can increase levels of proteins which cause cell cycle re-entry and cell death, leading to AD <a href="http://www.ncbi.nlm.nih.gov/pubmed/20436277" target="_blank">(Frade & Lopez-Sanchez 2010)</a>. BDNF can combat these effects. BDNF is a trophic factor, able to help sustain dying brain cells. It can alter connectivity, synapse strength and neurogenesis and so must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could more wildly change neuronal functions and circuits.
 +
</div>
 +
</div>
 +
 +
<div class="gap"></div>
 +
<p class="major_title">IP-10</p>
 +
<div class="row_small">
 +
<div class="part" style="background-image:url('http://2013.igem.org/wiki/images/6/64/IP10_png2.png');"></div>
 +
<div class="description">
 +
<p class="body_text"><b>
 +
This is a small non-inflammatory chemokine that induces chemotaxis [internal link to chemotaxis page] in macrophages. Microglia originate from a macrophage lineage. It elicits its effect through cell surface chemokine receptor CXCR3. Plaques already attract microglia, but this is partly due to local microglial activation. De-activated GEM will not produce many chemokines, so in order attract more GEM (as well as native microglia) to the plaque site, in order to speed up Aβ clearance.
 +
</div>
 +
</div>
<!-- END CONTENT ------------------------------------------------------------------------------------------------------>
<!-- END CONTENT ------------------------------------------------------------------------------------------------------>

Latest revision as of 23:18, 4 October 2013

CIRCUIT OVERVIEW

IGEM: Intelligently Genetically Engineered Microglia

Our ambitious project concerns bringing synthetic biology to the brain in order to try a novel approach to tackling Alzheimer’s Disease (AD). Microglia are mobile brain cells, making them an ideal chassis. To do this, our proposed treatment would involve extracting microglia from a patient, or using a specially bred immortalised line of human microglia, to avoid rejection, and transfecting it with our new genetic circuit. Alternatively, we could use nano-complexes to transfect native microglia. Implantation of genetically engineered microglia(GEM) or DNA into the brain would be performed via micro-neurosurgery. Our circuit is designed to detect amyloid plaques, attract other microglia, degrade the plaques, reduce neuroinflammation and support dying neurons. Theoretically, this should halt the progression of Alzheimer’s disease and could lead to other forms of neuro-genetic engineering.

Our GEM, selected for using zeocin resistance, would be injected into the key areas of pathology in the AD brain; the neocortex, limbic structures, hippocampus, amygdala, and some of the brainstem nuclei. Microglia are naturally drawn towards the senile plaques characteristic of AD. Senile plaques create free radicals, so as the GEM near the plaques oxidative stress increases. In response, our oxidative stress promoter will increase the transcription of key genes. Their protein products are matrix metalloproteinase 9 (MMP-9) , vasoactive intestinal peptide(VIP), brain derived neurotrophic factor (BDNF) and a chemoattractant called interferon gamma-induced protein 10(IP-10).

Brain cell death in AD is engendered by neuroinflammation due to microglia activation. VIP is able to de-activate GEM and native microglia. Activated microglia do not, however, produce proteases to clear plaques or attract other microglia. MMP-9 is capable of degrading β-amyloid fibrils and soluble β-amyloid in situ (Yan et al. 2006). Many theories of AD causation see amyloid build up as key to pathogenesis. Increasing clearance could therefore halt disease progression, and synthetically expressing MMP-9 in volume will ensure greater than natural plaque degradation even after microglial inactivation. In order attract more GEM to increase clearance rate, the chemoattractant is produced. AD may also arise due to insiufficient BDNF signaling (Frade & Lopez-Sanchez 2010), and BDNF is able to support dying neurons, which is why we also propose its production.

Zeocin Resistance

Zeocin is a glycopeptide antibiotic capable of killing most bacteria, fungi, yeast, plant, and animal cells by intercalating DNA and inducing double strand breakage. This makes zeocin resistance an ideal selective maker for our project, which involves both bacterial and mammalian chassis. The product of the ''Sh ble'' gene, isolated from the bacterium Streptoalloteichus hindustanus (Gatignol et al. 1988), confers zeocin resistance to transfected/transformed cells. Sh ble is a small binding protein with strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA.

Oxidative Stress Promoter

Oxidative stress via free radical production increases with proximity to senile plaques (Colton et al., 2000). The microglia immune response around plaques also increases oxidative stress. Therefore, we have designed a promoter which will initiate transcription in response to oxidative stress to ensure the production of key proteins only in the plaques’ locales. This promoter is an improvement of a yeast minimal promoter (cyc100) already in the registry. NF-κB is a transcription factor (pictured to the left) which translocates to the nucleus under oxidative stress (Shi et al., 2003), and binds to the sequence GGGAATTT (Park et al., 2009). Thus, by placing this site upstream of the yeast minimal promoter, we created a novel mammalian promoter which initiates transcription in response to oxidative stress.

Active MMP-9

MMP-9, also known as gelatinase B, is most commonly known for its role in breaking down the extracellular matrix. Naturally it is secreted in its inactive form and must be cleaved by other proteases, but our GEM are are meant to produce just the active form. It has been shown by Yan et al. that MMP-9 is the only known endogenous protease that degrades both fibrillar and soluble forms of amyloid-β peptide (Aβ). This satisfies the 'Amyloid Cascade Hypothesis' as well as theories that see plaques as neuroprotective, and soluble Aβ as the real threat. MMP-9 is expressed at low basal levels in microglia and may keep plaque size in dynamic equilibrium (Yan et al. 2006). Over producing it in (inactive) GEM could greatly improve both soluble and insoluble Aβ clearance. MMP-9 must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could cause damage to brain tissue if, for example, injected into the brain.

VIP

This is a hormone containing 28 amino acid residues. It is a powerful anti-inflammatory neuropeptide. It exhibits neuroprotection in AD model systems (Goze et al. 1996), able to save neurons exposed to excessive defective Aβ (70% success rate (Goze et al. 1996) by limiting neuroinflammation). VIP de-activates microglia (Delgado and Ganea 2003), inhibiting the production of inflammatory chemokines from microglia and reduces chemotaxis. VIP must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could inactivate too much of the brain immune if, for example, injected into the brain. If constitutively expressed, it would stop GEM migrating to plaques.

BDNF

While there is no change in BDNF levels in AD patients (O’Bryant et al. 2009), oxidative stress in older brains can increase levels of proteins which cause cell cycle re-entry and cell death, leading to AD (Frade & Lopez-Sanchez 2010). BDNF can combat these effects. BDNF is a trophic factor, able to help sustain dying brain cells. It can alter connectivity, synapse strength and neurogenesis and so must be delivered in GEM and expressed only in the vicinity of plaques, as otherwise it could more wildly change neuronal functions and circuits.

IP-10

This is a small non-inflammatory chemokine that induces chemotaxis [internal link to chemotaxis page] in macrophages. Microglia originate from a macrophage lineage. It elicits its effect through cell surface chemokine receptor CXCR3. Plaques already attract microglia, but this is partly due to local microglial activation. De-activated GEM will not produce many chemokines, so in order attract more GEM (as well as native microglia) to the plaque site, in order to speed up Aβ clearance.