Difference between revisions of "Team:UCopenhagen/Number-Control"

 
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                     <h2 class="section-heading">Introduction </h2>
 
                     <h2 class="section-heading">Introduction </h2>
 
                     <p class="lead">
 
                     <p class="lead">
The third mechanism that we have investigated in our project is protein import into bacterial cells, as a stand in for a symbiont. In the two best known endosymbiotic organelles, mitochondria and chloroplasts, a majority of gene expression has moved from the symbiont to the host. Because this relationship seems to be an evolutionary foundation of the known endosymbiotic relationships, we will attempt to imitate this concept.
+
 
<br><br>
+
The second mechanism we considered is <i>number control</i>. This is the intertwinement of the symbiont and host replication cycles. We believe this to be an imperative function of a healthy relationship between host and symbiont. In fact, the host cell is limited in size and it will be able to contain only a defined number of cells. Moreover, the exchange of nutrients also require a tightly regulated balance. For example, the mechanism of <i>interdependency</i> is based upon the providing of enough tryptophan to the host cell. This would not only depend on the production of amino acid per cell but also on the number of symbionts. Similarly, the symbionts won’t be able to directly access any resource not
Besides our interest in the evolutionary aspect of protein import, we believe it is valuable when approaching modular endosymbiosis. The principle of protein import would enable additional modularity in an endosymbiotic system, as one host can be manipulated to produce proteins to be utilized by a range of symbionts.  
+
provided by the host cell. Hence, as in the financial market, a correct balance of consumer and producer must be kept to avoid our symbiotic relationship turning into a parasitic relationship. Number control can be seen as the <i>New Deal</i> of endosymbiosis.</p> <br>
<br><br>
+
 
The majority of the proteins, destined for mitochondria, are expressed in the cytosol and subsequently imported across the membranes via transport complexes taking up unfolded peptides with an N-terminal signal sequence targeting them to the mitochondria (Schmidt <i>et al</i> 2010). Similar transport complexes are found in chloroplast. We want a similar targeted uptake, but in a simpler system. Thus, we utilise a small peptide shown to penetrate many types of cells; a Cell Penetrating Peptide (CPP) (Chang <i>et al</i> 2005, and Chang<i>et al</i> 2014).  
+
<p class="lead">Our vision is to use a modular system based on <b>C</b>lustered <b>R</b>egularly <b>I</b>nverted <b>S</b>hort <b>P</b>alindromic <b>R</b>epeats interference (CRISPRi) technology to efficiently connect the symbiont cell replication cycle to the status of the endosymbiosis relationship. As a first step towards an automated number control system, we will investigate the control of cell replication through DNA replication inhibition and model the incorporation of a cell density based system - that is, a quorum sensing circuit - regulating the replication control mechanism.</p>
 +
 
 +
 
 
</p>  
 
</p>  
 
                
 
                
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                     <h2 class="section-heading">Final Design </h2>
 
                     <h2 class="section-heading">Final Design </h2>
 
                                         <p class="lead">
 
                                         <p class="lead">
<strong>Background</strong>:  CPPs are small peptides, typically rich in arginines, which are able to facilitate transport of a wide variety of cargos across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain (Eudes and Chugh, 2008). In recent years, research on creating synthetics CPPs has been conducted and especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells. These experiments have shown that GFP connected to a CPP has entered the cells contained in vesicles (Chang <i>et al</i> 2005).  
+
<b>Background:</b> Prior to cell replication a bacteria need to double all cell components, including the genomic DNA. The DNA replication begins with the opening of the double strand DNA (dsDNA) carried out by the protein <i>helicase</i>. Preventing the attachment of helicase to the dsDNA will prevent double strand separation, genome replication, and ultimately, cell division.</p><br>
<br><br>
+
<p class="lead">To disrupt the helicase-DNA interaction, we designed a genetic regulation tool based on CRISPR interference (CRISPRi). This technology was first developed by Qi <i>et al</i> (2013) and is constituted by two basic blocks, i.e. a guide RNA (sgRNA) complementary to a target sequence and a catalytically dead Cas9 endonuclease (dCas9). dCas9 differentiate from Cas9 by its lack of nuclease activity. Hence, dCas9 will bind to the sgRNA and sit on the target DNA sequence, without altering permanently its structure. The presence of dCas9 on the target site sterically blocks the binding of other proteins, such as helicase.</p><br>
If CPP can be used as a protein tag for import into an endosymbiotic symbiont, the host proteins targeted to the symbiont would simply need the CPP added.  
+
<br><br>
+
<strong>Goal</strong>: Evaluate the efficiency of protein uptake by our <i>Escherichia coli</i> chassis in presence and absence of the cell penetrating peptide (CPP).  
+
<br><br>
+
  
<strong>Circuits and Biobricks</strong>: The parts in our circuit are fluorescent proteins and CPP. <br><br>
+
<p class="lead"><b>Our goal</b> is to inhibit attachment of helicase to the origin of replication (ORI), using CRISPRi system targeting the ORI, thus disrupting the cell replication of <i>E.coli</i>.</p><br>
We have chosen the yellow and blue fluorescent proteins (<a href="http://parts.igem.org/Part:BBa_K864100">YFP</a> and <a href="http://parts.igem.org/Part:BBa_K592100">BFP</a>) from the Biobrick repository, and improved them by adding the CPP sequence to the C-terminal end. <br><br>
+
 
YFP: <a href="http://parts.igem.org/Part:BBa_K2455002">BBa_K2455002 </a><br><br>
+
<p class="lead"><b>Circuits and biobricks:</b> Our CRISPRi system is composed by a catalytically dead Cas9 (dCas9) and three guide RNAs (sgRNAs) complementary to three distinct sites of <i>E.coli</i> origin of replication (OriC), i.e. on the DnaA helicase binding box (sgRNA1), before the DnaA binding box (sgRNA2) and after it (sgRNA3)(Wiktor et al., 2016). dCas9 is expressed from an anhydrotetracycline (aTc) inducible promoter and the sgRNAs are expressed separately from a minimal constitutive promoter (J23119). The choice of expressing the sgRNA constitutively arise from the higher efficiency of dCas9 to assemble with the sgRNA if the latter is already present in the cytosol (ref). The assembled protein-sgRNA will then bind to the complementary region on the bacterial chromosome and occupy that area. Detachment of dCas9-sgRNA complex from the target sequence can be achieved by incubating the cells at 42 °C (Wiktor et al., 2016) </p><br>
BFP: <a href="http://parts.igem.org/Part:BBa_K2455005">BBa_K2455005  </a> <br><br>
+
 
 +
<p class="lead">The <b>quorum sensing (QS) model</b> is based on the Rhl genetic circuit found in <i>Pseudomona aeruginosa</i>.</p>
  
Additionally, we created a biobrick of CPP with a USER casette, ready for insertion of any protein to be imported: <a href="http://parts.igem.org/Part:BBa_K2455003">BBa_K2455003</a>.
 
<br><br>
 
YFP and BFP were chosen to avoid overlapping colours with FM4-64, a red staining used for membranes, in case we wanted to look into the localization and potential vesicle breaking using confocal microscopy. As it turned out, we did not get far enough in the lab to do this.
 
 
</p>   
 
</p>   
 
         </div>
 
         </div>
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                     <h2 class="section-heading">Experiments</h2>
 
                     <h2 class="section-heading">Experiments</h2>
 
                     <p class="lead">
 
                     <p class="lead">
 
 
<h4>Overview</h4>
 
<h4>Overview</h4>
<br>
 
<p class="lead">
 
General verification</p>
 
<p class="lead">
 
Vector creation
 
 
<ul style="text-align:left; color:white;">
 
<ul style="text-align:left; color:white;">
<li>Biobrick compatible (failed) </li>
+
<li>General procedure</li>
<li>Vector design </li>
+
<li>Creation of working <i>E. coli</i> DH5-α strains:</li>
<li>CPP tag insertion </li>
+
<li>OD600 growth curve</li>
 
</ul>
 
</ul>
 
</p>
 
</p>
<br>
 
 
<p class="lead">
 
<p class="lead">
Evaluating protein import:
+
<h4>General procedure</h4>
 +
<p class="lead">To obtain large quantities of our plasmids 3xFLAG-pdCas9 and pgRNA-bacteria we transformed them into <i>E. coli</i> mach1 using Mix&Go (Zymo Research). To verify each transformation we purified the plasmid with column purification, linearized with proper restriction enzyme (RE) digestion to avoid supercoiling, and ran the linearized product in 1% agarose gel electrophoresis. <br><br>
 +
Moreover, all the purified plasmids were sent for sequencing (Macrogen EZ-seq commercial service).</p></p>
 +
<p class="lead">
 +
<h4>Creation of working <i>E. coli</i> DH5-α strains</h4>
 +
<p class="lead">
 +
The following commercially available plasmids from AddGene were selected for our experiment:
 
<ul style="text-align:left; color:white;">
 
<ul style="text-align:left; color:white;">
<li>Expression of fluorescent proteins</li>
+
<li>3xFLAG-dCas9/p-bacteria (Addgene #64325)</li>
<li>Purification and import of the expressed proteins</li>
+
<li>pgRNA-bacteria (Addgene #44251)</li></ul></p>
</ul>
+
</p>
+
 
<br>
 
<br>
<h4>Verifications and biobrick creation</h4>
 
  
<p class="lead">
+
<figure>
 
+
<br>
In all three sub projects, we have used gel electrophoresis and sequencing to verify our stepwise experiments. Read more about our general verifications and biobrick creation under <a href="https://2017.igem.org/Team:UCopenhagen/Interdependency">interdependency</a>.
+
                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/7/76/Noctrl_dCas9_plasmid.png" alt="" width="250" height="200">
</p>
+
<br>
 
+
<figcaption><b>Figure 1 </b>Plasmid: 3xFLAG-dCas9/p-bacteria (Addgene #64325)
<h4>Vector creation</h4>
+
</figcaption>
 
+
</figure>
<p class="lead">
+
 
+
Our vector is a modified version of the <a href="https://www.addgene.org/vector-database/2623/">pET102 vector</a>, which contains a USER cassette, and a his tag after the USER cassette. The USER cassette is used for cloning genes into. We build the vector design on prSET102; an existing vector in our lab, containing the USER cassette and some restriction sites. <br><br>  
+
We have made two versions of the vector: one for expression of untagged YFP and BFP, that was also used in the interdependency subproject. The other has a CPP tag before the USER cassette, which will add CPP to the proteins.
+
 
+
</p>
+
  
 
<figure>
 
<figure>
 
<br>
 
<br>
                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/5/5d/CPPvector.png" alt="" width="250" height="200">
+
                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/5/5e/Noctrl_pgRNA_plasmid.png" alt="" width="250" height="200">
 
<br>
 
<br>
  <figcaption><b>Figure 1 </b>Overview of final vectors. First level: The two vectors, unlinearized. Second level: linearized by opening in USER casette. Third level: Insertion of YFP. 4: Produced YFP protein with and without CPP tag. YFP used as example; the same method is used for expression of BFP with and without CPP tag.
+
  <figcaption><b>Figure 2 </b>Plasmid: pgRNA-bacteria (Addgene #44251) which was used for insertion of seed sequence
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
 +
 
<br>
 
<br>
 +
<p class="lead">
 +
 +
The three seed sequences designed were inserted using PCR. Thus creating the new plasmids pgRNA1, pgRNA2, and pgRNA3.<br><br>
 +
We used the following primers for insertion of the seed sequences, where the underlined sequence is SpeI restriction site and the bold text is the seed sequence complementary to the E. coli DH5-α OriC.
 +
 +
<ul style="text-align:left; color:white;">
 +
<li>fw.sgRNA1 CCACTAGTGCACTGCCCTGTGGATAACAGTTTTAGAGCTAGAAATAGCAAG</li>
 +
<li>fw.sgRNA2 CCACTAGTTTGAGAAAGACCTGGGATCCGTTTTAGAGCTAGAAATAGCAAG</li>
 +
<li>fw.sgRNA3 CCACTAGTGATCATTAACTGTGAATGATGTTTTAGAGCTAGAAATAGCAAG</li>
 +
<li>rv.sgRNA GGACTAGTATTATACCTAGGACTGAG</li></ol></p>
 +
 +
<p class="lead">After purification of the modified pgRNA plasmids, transformation of <i>E. coli</i> DH5-α was carried out using heat-shock protocol. And the following strains were obtained:<ul style="text-align:left; color:white;">
 +
<li><i>E. coli</i> DH5-α pdCas9;</li>
 +
<li><i>E. coli</i> DH5-α pgRNA1;</li>
 +
<li><i>E. coli</i> DH5-α pgRNA2;</li>
 +
<li><i>E. coli</i> DH5-α pgRNA3;</li>
 +
<li><i>E. coli</i> DH5-α pdCas9 pgRNA1;</li>
 +
<li><i>E. coli</i> DH5-α pdCas9 pgRNA2;</li>
 +
<li><i>E. coli</i> DH5-α pdCas9 pgRNA3.</li></ul></p>
 +
 +
<h4>Growth curve (OD<sub>600</sub>)</h4>
 +
<p class="lead">The growth rate of the obtained strains was investigated in absence and presence of the dCas9 inductor tetracycline (tet) using absorbance measurements (protocol).<br><br>
 +
The strains <i>E. coli</i> DH5-α pdCas9, <i>E. coli</i> DH5-α pgRNA1, <i>E. coli</i> DH5-α pgRNA2, and <i>E. coli</i> DH5-α pgRNA3 cannot be considered proper negative control since they are cultivated with different antibiotics. Hence, three new negative control strain were designed and created:
 +
 +
<ul style="text-align:left; color:white;">
 +
<li><i>E. coli</i> DH5-α empty pdCas9 (EpdCas9). That is, the pdCas9 plasmid with truncated dCas9 CDS using EcoRI restriction);</li>
 +
<li><i>E. coli</i> DH5-α pdCas9 + pgRNA-bacteria (that is, the sgRNA scaffold without seed sequence);</li>
 +
<li><i>E. coli</i> DH5-α EpdCas9 pgRNA-bacteria (we did manage to obtain this strain).</li></ul>
 +
</p>
 +
<p class="lead">Moreover, considering that the stop of DNA replication is expected to cause cell enlargement (Wiktor <i>et al</i>., 2016) the OD600­ measurement results might altered by the cell size. To overcome this and other issues a new protocol was design.<br><br>
 +
However, the difference in growth observed was never significant. Hence, we decided to focus on the numerical model describing the <i>Number Control</i> project, where we also explored the integration of a quorum sensing circuit.</p>
  
 
</div>
 
</div>
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<div class="container">
 
<div class="container">
 
             <div>
 
             <div>
                    <hr class="section-heading-spacer">
 
 
                     <div class="clearfix"></div>
 
                     <div class="clearfix"></div>
 
                     <h2 class="section-heading">Design process/future</h2>
 
                     <h2 class="section-heading">Design process/future</h2>
 +
<p class="lead">After the design of the growth rate experiment, we started exploring other possible layers of complexity. Namely, Wiktor <i>et al</i>. (2016) has shown that upon successful genome replication arrest, the cell stays metabolically active, accumulating biomass. For this reason, we suggest the inhibition of lipid biosynthesis to limit cell membrane growth, hence cell enlargement. This could be obtained with a similar sgRNA-dCas9 circuit targeting the gene fabI, CDS for the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR). This protein is in fact essential for the type II fatty acid biosynthesis and is commonly target of bacteriostatic drugs (Escaich, ‎2011).<br><br>
 +
Besides the autoinhibitory control of cell cycle, we are deeply interested in connecting the number control circuit to the host life cycle to further stabilize the relationship between host and symbiont. To do so, we propose to place the pgRNA-dCas9 system under control of three host-symbiont signals, i.e. symbiont abundance (that is, a quorum sensing sub system), host cell starvation, and host cell replication. Namely, a high symbiont abundance and/or a starvation status of the host cell would activate the dCas9 expression lowering or stopping symbiont replication. While the host cell initiation of replication would inactivate the dCas9 expression, so to allow an easy propagation of the symbiont.</p>
  
 
                   </div>
 
                   </div>

Latest revision as of 02:47, 2 November 2017

N U M B E R   C O N T R O L


Introduction

The second mechanism we considered is number control. This is the intertwinement of the symbiont and host replication cycles. We believe this to be an imperative function of a healthy relationship between host and symbiont. In fact, the host cell is limited in size and it will be able to contain only a defined number of cells. Moreover, the exchange of nutrients also require a tightly regulated balance. For example, the mechanism of interdependency is based upon the providing of enough tryptophan to the host cell. This would not only depend on the production of amino acid per cell but also on the number of symbionts. Similarly, the symbionts won’t be able to directly access any resource not provided by the host cell. Hence, as in the financial market, a correct balance of consumer and producer must be kept to avoid our symbiotic relationship turning into a parasitic relationship. Number control can be seen as the New Deal of endosymbiosis.


Our vision is to use a modular system based on Clustered Regularly Inverted Short Palindromic Repeats interference (CRISPRi) technology to efficiently connect the symbiont cell replication cycle to the status of the endosymbiosis relationship. As a first step towards an automated number control system, we will investigate the control of cell replication through DNA replication inhibition and model the incorporation of a cell density based system - that is, a quorum sensing circuit - regulating the replication control mechanism.

Final Design

Background: Prior to cell replication a bacteria need to double all cell components, including the genomic DNA. The DNA replication begins with the opening of the double strand DNA (dsDNA) carried out by the protein helicase. Preventing the attachment of helicase to the dsDNA will prevent double strand separation, genome replication, and ultimately, cell division.


To disrupt the helicase-DNA interaction, we designed a genetic regulation tool based on CRISPR interference (CRISPRi). This technology was first developed by Qi et al (2013) and is constituted by two basic blocks, i.e. a guide RNA (sgRNA) complementary to a target sequence and a catalytically dead Cas9 endonuclease (dCas9). dCas9 differentiate from Cas9 by its lack of nuclease activity. Hence, dCas9 will bind to the sgRNA and sit on the target DNA sequence, without altering permanently its structure. The presence of dCas9 on the target site sterically blocks the binding of other proteins, such as helicase.


Our goal is to inhibit attachment of helicase to the origin of replication (ORI), using CRISPRi system targeting the ORI, thus disrupting the cell replication of E.coli.


Circuits and biobricks: Our CRISPRi system is composed by a catalytically dead Cas9 (dCas9) and three guide RNAs (sgRNAs) complementary to three distinct sites of E.coli origin of replication (OriC), i.e. on the DnaA helicase binding box (sgRNA1), before the DnaA binding box (sgRNA2) and after it (sgRNA3)(Wiktor et al., 2016). dCas9 is expressed from an anhydrotetracycline (aTc) inducible promoter and the sgRNAs are expressed separately from a minimal constitutive promoter (J23119). The choice of expressing the sgRNA constitutively arise from the higher efficiency of dCas9 to assemble with the sgRNA if the latter is already present in the cytosol (ref). The assembled protein-sgRNA will then bind to the complementary region on the bacterial chromosome and occupy that area. Detachment of dCas9-sgRNA complex from the target sequence can be achieved by incubating the cells at 42 °C (Wiktor et al., 2016)


The quorum sensing (QS) model is based on the Rhl genetic circuit found in Pseudomona aeruginosa.


Experiments

Overview

  • General procedure
  • Creation of working E. coli DH5-α strains:
  • OD600 growth curve

General procedure

To obtain large quantities of our plasmids 3xFLAG-pdCas9 and pgRNA-bacteria we transformed them into E. coli mach1 using Mix&Go (Zymo Research). To verify each transformation we purified the plasmid with column purification, linearized with proper restriction enzyme (RE) digestion to avoid supercoiling, and ran the linearized product in 1% agarose gel electrophoresis.

Moreover, all the purified plasmids were sent for sequencing (Macrogen EZ-seq commercial service).

Creation of working E. coli DH5-α strains

The following commercially available plasmids from AddGene were selected for our experiment:

  • 3xFLAG-dCas9/p-bacteria (Addgene #64325)
  • pgRNA-bacteria (Addgene #44251)




Figure 1 Plasmid: 3xFLAG-dCas9/p-bacteria (Addgene #64325)


Figure 2 Plasmid: pgRNA-bacteria (Addgene #44251) which was used for insertion of seed sequence

The three seed sequences designed were inserted using PCR. Thus creating the new plasmids pgRNA1, pgRNA2, and pgRNA3.

We used the following primers for insertion of the seed sequences, where the underlined sequence is SpeI restriction site and the bold text is the seed sequence complementary to the E. coli DH5-α OriC.

  • fw.sgRNA1 CCACTAGTGCACTGCCCTGTGGATAACAGTTTTAGAGCTAGAAATAGCAAG
  • fw.sgRNA2 CCACTAGTTTGAGAAAGACCTGGGATCCGTTTTAGAGCTAGAAATAGCAAG
  • fw.sgRNA3 CCACTAGTGATCATTAACTGTGAATGATGTTTTAGAGCTAGAAATAGCAAG
  • rv.sgRNA GGACTAGTATTATACCTAGGACTGAG
  • After purification of the modified pgRNA plasmids, transformation of E. coli DH5-α was carried out using heat-shock protocol. And the following strains were obtained:

    • E. coli DH5-α pdCas9;
    • E. coli DH5-α pgRNA1;
    • E. coli DH5-α pgRNA2;
    • E. coli DH5-α pgRNA3;
    • E. coli DH5-α pdCas9 pgRNA1;
    • E. coli DH5-α pdCas9 pgRNA2;
    • E. coli DH5-α pdCas9 pgRNA3.

    Growth curve (OD600)

    The growth rate of the obtained strains was investigated in absence and presence of the dCas9 inductor tetracycline (tet) using absorbance measurements (protocol).

    The strains E. coli DH5-α pdCas9, E. coli DH5-α pgRNA1, E. coli DH5-α pgRNA2, and E. coli DH5-α pgRNA3 cannot be considered proper negative control since they are cultivated with different antibiotics. Hence, three new negative control strain were designed and created:

    • E. coli DH5-α empty pdCas9 (EpdCas9). That is, the pdCas9 plasmid with truncated dCas9 CDS using EcoRI restriction);
    • E. coli DH5-α pdCas9 + pgRNA-bacteria (that is, the sgRNA scaffold without seed sequence);
    • E. coli DH5-α EpdCas9 pgRNA-bacteria (we did manage to obtain this strain).

    Moreover, considering that the stop of DNA replication is expected to cause cell enlargement (Wiktor et al., 2016) the OD600­ measurement results might altered by the cell size. To overcome this and other issues a new protocol was design.

    However, the difference in growth observed was never significant. Hence, we decided to focus on the numerical model describing the Number Control project, where we also explored the integration of a quorum sensing circuit.

Design process/future

After the design of the growth rate experiment, we started exploring other possible layers of complexity. Namely, Wiktor et al. (2016) has shown that upon successful genome replication arrest, the cell stays metabolically active, accumulating biomass. For this reason, we suggest the inhibition of lipid biosynthesis to limit cell membrane growth, hence cell enlargement. This could be obtained with a similar sgRNA-dCas9 circuit targeting the gene fabI, CDS for the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR). This protein is in fact essential for the type II fatty acid biosynthesis and is commonly target of bacteriostatic drugs (Escaich, ‎2011).

Besides the autoinhibitory control of cell cycle, we are deeply interested in connecting the number control circuit to the host life cycle to further stabilize the relationship between host and symbiont. To do so, we propose to place the pgRNA-dCas9 system under control of three host-symbiont signals, i.e. symbiont abundance (that is, a quorum sensing sub system), host cell starvation, and host cell replication. Namely, a high symbiont abundance and/or a starvation status of the host cell would activate the dCas9 expression lowering or stopping symbiont replication. While the host cell initiation of replication would inactivate the dCas9 expression, so to allow an easy propagation of the symbiont.


References

Find Incell here: