Team:Tokyo-NoKoGen/Project/aggregation

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<h2>Introduction</h2>
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In our EcoTanker, the goal is to collect objective substance automatically by using <i>E. coli</i>. However, to collect substance, we have to collect the cell at first. So we aimed to construct a device, which signals <i>E. coli</i> to self-aggregate and we focused on a protein, Antigen 43. This protein is derived from <i>E. coli</i>.
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Antigen 43 consists of two protein subunits, α and β, with apparent molecular masses of about 50 and 53 kDa. The β subunit is attached to the cell surface via interaction with the β subunit, which is an integral outer membrane component (Fig. 1). Antigen 43 has the N-terminal signal peptide and it directs translocation across the cytoplasmic membrane to the periplasm via the general secretory pathway. Subsequently, the β domain forms a β barrel structure in the outer membrane through which the β domain gains access to the cell exterior [1].
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<h2>Why is this device needed?</h2>
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Aggregation device is needed because
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<ul>Taking up a target material in <i>E. coli</i>.</ul>
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<ul>Self-aggregation of <i>E. coli</i>. : Skipping the harvest work.</ul>
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<h2>What is this device composed inside it?</h2>
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This device expresses Antigen 43, which is encoded by the gene agn43. For the promoter, we used OmpR (+) promoter (ompC promoter) (BBa_R0082)(Fig. 2). This promoter responds to phosphorylated OmpR (response regulator). OmpR will phosphorylated by EnvZ, which is a histidine kinase.
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Overview
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<h2>How does this device work in EcoTanker?</h2>
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In our EcoTanker, the goal is to collect objective substance automatically by using E. coli. However, to collect substance, we have to collect the cell at first.  
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<img src="https://static.igem.org/mediawiki/2010/4/4c/Aggregation_Fig3.JPG" alt="" style="float:right" width="300"/>
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So we aimed to construct a device, which signals E. coli to self-aggregate and we focused on a protein, Antigen 43. This protein is derived from E. coli.  
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This device will be activated by the Green light receptor (detail is on the page of PHOTOCONTROL). When we want to collect <i>E. coli</i>, we will flash green light and aggregate it (Fig. 3).
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<h2>Progress</h2>
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<p>The wild type of Antigen 43 contains 6 <i>Pst</i>I sites. So, to use as a BioBrick, we had to modify this 6 PstI sites. To modify, we choose Overlap PCR and the scheme will be below. First, we amplify 3 fragments by using the genome as a template and primers to modify the PstI sites. Then, by using these 3 fragments as a primer, and the forward and reverse primer when we cloned Antigen 43, we amplified 4 fragments. And we performed overlap PCR with this 4 fragments by using the forward and reverse primer. We confirmed the amplification of the gene of Antigen 43. After digesting this fragment with EcoRI and PstI, ligated to pSB1C3 to construct the plasmid pSB1C3-Antigen 43(BBa_K317008). We analysis the sequence and confirmed that Antigen 43 didn’t have any PstI sites. By using this new BioBrick, we constructed transcriptional unit, generator, and 4 devices. We also submitted these 6 parts as a new BioBrick(Table).</p><br clear="right">
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Method and Result
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We performed TA-cloning against the gene of Antigen 43 from the E. coli K-12 genome. At this time, we added the BioBrick prefix and suffix by using primers. After digesting the PCR product with EcoRI and SpeI, ligated the PCR product to pGEM-T easy vector and transformed E. coli DH5. Then, extracted the plasmid and analyze the sequence to confirm the success in cloning Antigen 43 gene. From sequence analysis, confirmed a plasmid that harbored the gene of Antigen 43 without any mutations. After confirming the amplification oft the objective sequence, digested the plasmid(pGEM-T easy vector – Antigen 43) with EcoRI and SpeI, ligated to the BioBrick vector, pSB1C3.  
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We confirmed the function of Antigen 43 by using 2 devices that expresses Antigen 43 constitutively and evaluate by measuring OD660 approximately 1 cm from the top [1].
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The methods will be, inoculate a singe colony to 5 ml of LB medium and incubate at 37℃ over night. Then, stopped incubation and stand the culture solution. Then, measured the OD660 from the top of 1 cm of the culture. The results of measuring OD660 (Fig. 5) and a picture of appearance of E. coli sinking will be shown below (Fig.5).
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From the results, the <i>E. coli</i> that harbors the plasmids (Constitutive promoter-RBS-Antigen 43-Double terminator) show decreasing of OD660 although the strength of the promoter is weak (BBa_J23102 is strong and BBa_J23109 is weak). So, we can say low expression of Antigen 43 will be enough to sink the <i>E. coli</i>.</p>
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The wild type of Antigen 43 contains 6 PstI sites. So, to use as a BioBrick, we had to modify this 6 PstI sites. To modify, we choose Overlap PCR and the scheme will be below. First, we amplify 3 fragments by using the genome as a template and primers to modify the PstI sites. Then, by using these 3 fragments as a primer, and the forward and reverse primer when we cloned Antigen 43, we amplified 4 fragments. And we performed overlap PCR with this 4 fragments by using the forward and reverse primer. We confirmed the amplification of the gene of Antigen 43. After digesting this fragment with EcoRI and PstI, ligated to pSB1C3 to construct the plasmid pSB1C3-Antigen 43(BBa_K317008). We analysis the sequence and confirmed that Antigen 43 didn’t have any PstI sites. By using this new BioBrick, we constructed transcriptional unit, generator, and 4 devices. We also submitted these 6 parts as a new BioBrick(Table).
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Table. New BioBrick parts constructed by Aggregation team
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<img src="https://static.igem.org/mediawiki/2010/6/6b/Aggrigation_Fig4.gif" alt="" width="450px" style="float:left" />
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BBa_K317007 Antigen 43(Wild type)
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<th><img src="https://static.igem.org/mediawiki/2010/0/01/Aggregation_Fig5.JPG" alt="" width="300px" height="400px" />
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BBa_K317008 Antigen 43
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BBa_K317018 RBS-Antigen 43
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BBa_K317023 RBS-Antigen 43-Double terminator
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BBa_K317024 Constitutive promoter(BBa_J23102)-RBS-Antigen 43-Double terminator
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BBa_K317025 Constitutive promoter(BBa_J23107)-RBS-Antigen 43-Double terminator
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BBa_K317026 Constitutive promoter(BBa_J23109)-RBS-Antigen 43-Double terminator
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BBa_K317027 OmpR positive promoter(BBa_R0082)- RBS-Antigen 43-Double terminator
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We confirmed the function of Antigen 43 by using 2 devices that expresses Antigen 43 constitutively and evaluate by measuring OD660.
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<br clear="left" />
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The methods will be, inoculate a singe colony to 5 ml of LB medium and incubate at 37℃ over night. Then, stoped incubation and stand the culture solution. Then, measured the OD660 from the top of 1 cm of the culture. The results of measuring OD660 and a picture of appearance of E. coli sinking will be shown below(Fig.1).  
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<p>BioBricks we submitted are bellows.</p>
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From the results, the E. coli which harbor the plasmids(Constitutive promoter-RBS-Antigen 43-Double terminator) shows decreasing of OD660 although the strength of the promoter is weak(BBa_J23102 is strong and BBa_J23109 is weak). So, we can say low expression of Antigen 43 will be enough to sink the E. coli.  
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<img src="https://static.igem.org/mediawiki/2010/d/da/Aggregation_table.png" alt="" width="90%" />
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<img src="https://static.igem.org/mediawiki/2010/9/94/Aggregation_fig_1.tif">  
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<h2>References</h2>
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Fig.1 Evaluation of the function of Antigen 43 by measuring OD660
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<p>
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[1] KRISTIAN KJÆRGAARD, MARK A. SCHEMBRI, HENRIK HASMAN, AND PER KLEMM.
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Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens
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JOURNAL OF BACTERIOLOGY, 2000, p. 4789–4796, Vol. 182, No. 17</p>
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Latest revision as of 03:13, 28 October 2010

Introduction

broken
In our EcoTanker, the goal is to collect objective substance automatically by using E. coli. However, to collect substance, we have to collect the cell at first. So we aimed to construct a device, which signals E. coli to self-aggregate and we focused on a protein, Antigen 43. This protein is derived from E. coli. Antigen 43 consists of two protein subunits, α and β, with apparent molecular masses of about 50 and 53 kDa. The β subunit is attached to the cell surface via interaction with the β subunit, which is an integral outer membrane component (Fig. 1). Antigen 43 has the N-terminal signal peptide and it directs translocation across the cytoplasmic membrane to the periplasm via the general secretory pathway. Subsequently, the β domain forms a β barrel structure in the outer membrane through which the β domain gains access to the cell exterior [1].

Why is this device needed?

Aggregation device is needed because
    Taking up a target material in E. coli.
    Self-aggregation of E. coli. : Skipping the harvest work.

What is this device composed inside it?

This device expresses Antigen 43, which is encoded by the gene agn43. For the promoter, we used OmpR (+) promoter (ompC promoter) (BBa_R0082)(Fig. 2). This promoter responds to phosphorylated OmpR (response regulator). OmpR will phosphorylated by EnvZ, which is a histidine kinase.

How does this device work in EcoTanker?

This device will be activated by the Green light receptor (detail is on the page of PHOTOCONTROL). When we want to collect E. coli, we will flash green light and aggregate it (Fig. 3).

Progress

The wild type of Antigen 43 contains 6 PstI sites. So, to use as a BioBrick, we had to modify this 6 PstI sites. To modify, we choose Overlap PCR and the scheme will be below. First, we amplify 3 fragments by using the genome as a template and primers to modify the PstI sites. Then, by using these 3 fragments as a primer, and the forward and reverse primer when we cloned Antigen 43, we amplified 4 fragments. And we performed overlap PCR with this 4 fragments by using the forward and reverse primer. We confirmed the amplification of the gene of Antigen 43. After digesting this fragment with EcoRI and PstI, ligated to pSB1C3 to construct the plasmid pSB1C3-Antigen 43(BBa_K317008). We analysis the sequence and confirmed that Antigen 43 didn’t have any PstI sites. By using this new BioBrick, we constructed transcriptional unit, generator, and 4 devices. We also submitted these 6 parts as a new BioBrick(Table).


We confirmed the function of Antigen 43 by using 2 devices that expresses Antigen 43 constitutively and evaluate by measuring OD660 approximately 1 cm from the top [1]. The methods will be, inoculate a singe colony to 5 ml of LB medium and incubate at 37℃ over night. Then, stopped incubation and stand the culture solution. Then, measured the OD660 from the top of 1 cm of the culture. The results of measuring OD660 (Fig. 5) and a picture of appearance of E. coli sinking will be shown below (Fig.5). From the results, the E. coli that harbors the plasmids (Constitutive promoter-RBS-Antigen 43-Double terminator) show decreasing of OD660 although the strength of the promoter is weak (BBa_J23102 is strong and BBa_J23109 is weak). So, we can say low expression of Antigen 43 will be enough to sink the E. coli.


BioBricks we submitted are bellows.

References

[1] KRISTIAN KJÆRGAARD, MARK A. SCHEMBRI, HENRIK HASMAN, AND PER KLEMM. Antigen 43 from Escherichia coli Induces Inter- and Intraspecies Cell Aggregation and Changes in Colony Morphology of Pseudomonas fluorescens JOURNAL OF BACTERIOLOGY, 2000, p. 4789–4796, Vol. 182, No. 17

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