Template:Kyoto/Project/FlowerFairy

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===Realizing Flower Fairy in real world===
===Realizing Flower Fairy in real world===
[[File:FFT_kyoto.png‎ |left|300px]]
[[File:FFT_kyoto.png‎ |left|300px]]
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Have you ever seen flower fairies? Probably the answer is, "NO" (though some of you might have come across them in your childhood), because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition to the happy feelings, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. That is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!
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Have you ever seen flower fairies? Probably the answer is "NO", (though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. This is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!!
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In order to realize our dream, we focused on FT protein, known as Florigen.This protein is a kind of plant hormone. First, FT protein is produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.
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In order to realize our dream, we focused on '''FT protein''', known as Florigen.This protein is a kind of plant hormones. First of all, FT protein is produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.
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===Our Goal is to induce flower formation just by putting Flower Fairy ''E.coli'' on leaves!===
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===Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!===
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When you want to use our Flower Fairy ''E.coli'', all you have to do is just put them on plant leaves! When you spread Flower Fairy ''E.coli'', FT protein is secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br>
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When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT protein is secreted and penetrate the cell membrane of a plant, and the plant starts blooming.<br><br>
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<html>We had to go through four steps in order to achieve our goal――Flower Fairy <i>E.coli</i>.<br>
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<html>We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.<br>
These four steps are <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,
These four steps are <a href="#Expression">“<b>EXPRESSION</b>”</a>,<a href="#Secretion">”<b>SECRETION</b>”</a>,
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html>
<a href="#Penetration">”<b>PENETRATION</b>”</a>, and <a href="#Activation">”<b>ACTIVATION</b>”</a><br><br></html>
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<html>In each step, we have some problems to be attacked.<br>
<html>In each step, we have some problems to be attacked.<br>
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<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> (prokaryote) can express FT protein, because FT protein is derived from plant cells (eukaryote).<br>
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<a href="#Expression">“<b>EXPRESSION</b>”</a>; It is unclear whether <i>E.coli</i> can express FT protein, because FT protein is derived from plant cells .<br>
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<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, FT protein has to get out of the <i>E.coli</i>.<br>
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<a href="#Secretion">”<b>SECRETION</b>”</a>; After produced, ''E.coli'' have to secrete FT protein outside of themselves.<br>
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br>
<a href="#Penetration">”<b>PENETRATION</b>”</a>; FT protein has to penetrate into plant cells.<br>
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<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is not clear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html>
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<a href="#Activation">”<b>ACTIVATION</b>”</a>; Even if FT protein could get into the cells, it is unclear whether FT protein produced by <i>E.coli</i> can activate genes in shoot apex cells and induce flower formation.</html>
<html><a id="FFEResults"></a></html>
<html><a id="FFEResults"></a></html>
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=== Modifying ''FT'' gene for Biobrick ===
=== Modifying ''FT'' gene for Biobrick ===
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''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new biobrick parts of ''FT'' (Fig.1-1 C).  
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''FT'' gene is derived from ''Arabidopsis thaliana'', a model plant. Professor Araki in Kyoto University kindly gave us ''FT'' cDNA in TOPO blunt end 2(Invitrogen). ''FT'' cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified ''FT'' gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to ''FT''. Finally, we could make new BioBrick parts of ''FT'' (Fig.1-1 C).  
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]
[[File:FTabc.png|thumb|left|650px|Fig.1-1<br>A : Necessity for mutation of ''FT'' cDNA. ''FT'' sequence had two cleavage sites of iGEM restriction enzymes.<br>B : Inverse PCR Method<br> Inverse PCR is a measure of mutating ''FT'' gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.<br>C : Standardization of ''FT'' cDNA. We had mutated and added prefix and suffix to ''FT''.]]
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=== Confirming expression of FT ===
=== Confirming expression of FT ===
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====Proteins are required to go through two membranes====
====Proteins are required to go through two membranes====
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[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT proteins have two hurdles in order to get out of the cell]]
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[[File:Sec-intro-hurdles.png|thumb|right|350px|Fig.2-1 FT protein have two hurdles in order to get out of the cell]]
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''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', therefore, FT protein has to pass through the two membranes. So we searched for secretion systems to make FT proteins go through these membranes.  
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''E.coli'' have two membranes: inner membrane and outer membrane. To transport FT protein to outside of ''E.coli'', FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes.  
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====TorA signal enables proteins to go through the inner membrane via tat pathway====
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====TorA signal enables proteins to go through the inner membrane via Tat pathway====
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[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with torA signal go through the inner membrane to the periplasm.]]
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[[File:Tat pathway.png|thumb|right|250px|Fig.2-2 FT protein with TorA signal go through the inner membrane to the periplasm.]]
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Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation(Tat) pathway. Tat pathway is more suitable than other secretion systems for us, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is  the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and transports into the periplasm only proteins that have a TorA signal at their N terminals.
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Wild ''E.coli'' have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway ''E.coli'' secrete proteins into the periplasm keeping proteins’ conformation and function. The following is  the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm. <br clear="both"/>
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====We improved usability of TorA signal biobrick====
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====We improved usability of TorA signal BioBrick====
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We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS, which regulate additional processing time for iGEMers who use the parts. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, therefore, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, but TorA would be expressed. <br>
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We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as [http://partsregistry.org/Part:BBa_K638402 BBa_K638402]) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed. <br>
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br>
To solve these problems, we produced new applicable TorA signal [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] (Fig.2-2). Our part has two improvements. [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced [http://partsregistry.org/Part:BBa_K797002 BBa_K797002] and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly. <br>
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[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal biobricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]
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[[File:Sec-inner-torA2.png|thumb|center|600px|Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)]]
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]
[[File:Sec-ecoli-gfp.png|thumb|center|600px|Fig.2-4 ''E.coli'' (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. ''E.coli'' (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.]]
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We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''.  
We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of ''E.coli''. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of ''E.coli''.  
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'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' to secrete FT protein without cell lysis!!(Fig.2-7)'''
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'''We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make ''E.coli'' secrete FT protein without cell lysis!!(Fig.2-7)'''
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.
[[File:Sec-kill-assay2.png|thumb|center|700px|Fig.2-6 The result of evaluation of kil protein.
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And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]
And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.]]
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[[File:Sec-integration.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.
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[[File:Detail explanation of TAT system.png|thumb|center|600px|Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.
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  1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]
  1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.]]
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====Improvement of secretion system====
====Improvement of secretion system====
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Now we can enable'' E.coli'' to secrete FT proteins to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT proteins when they use Tat transporters which they inherently have.  
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Now we can let'' E.coli'' to secrete FT protein to the outside by using Tat pathway and kil protein. But ''E.coli'' secrete only a small amount of FT protein when they use Tat transporters which they inherently have.  
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To make E.coli secrete enough amount of FT proteins, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.
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To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild ''E.coli'' have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H<sup>+</sup> concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion.
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8)  
We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8)  
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9)  
This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make ''E.coli'' secrete more proteins with TorA.(Fig.2-9)  
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<gallery widths=300px heights=130px>  
<gallery widths=300px heights=130px>  
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)
File:Sec-futurework-construction.png|Fig.2-8 Tat secretion cassette; Tat ABC, pspA and constitutive promoter(BBa_K797004)
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File:Sec-futurework-pspA.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.
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File:Detail explanation of TAT system improvement.png|Fig.2-9 pspA helps proteins to go through Tat secretion pathway.
</gallery>  
</gallery>  
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The third step is '''PENETRATION'''.
The third step is '''PENETRATION'''.
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In order to induce flower formation, FT proteins from ''E.coli'' must enter into plant cells. This is because FT proteins upregulate other proteins which lead to flower formation in plant cells.
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In order to induce flower formation, FT protein from ''E.coli'' must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.
However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells.
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Secondly, the cell responds to this stimulus and starts to endocytose.
Secondly, the cell responds to this stimulus and starts to endocytose.
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Finally, proteins around the endocytosing region of the cell is taken into the cell.<br>
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Finally, proteins around the endocytosing region of the cell are taken into the cell.<br>
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R9 peptides seem to work effectively regardless of whether or not R9 peptides are fused with target proteins.  
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R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins.  
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br>
However, there are few examples about endocytosis of plants, so '''we needed to check the function of R9 when used on plant cells'''.<br>
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[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2  Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT proteins around an invaginating region of the cell are taken into the cell. ]]
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[[File:Pene-R9-mechanism.png|thumb|left|650px|Fig.3-2  Mechanism of endocytosis by R9 peptide <br> 1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.<br> 2.The cell responds to this stimulus and starts to endocytose.<br> 3.FT protein around an invaginating region of the cell are taken into the cell. ]]
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==== From the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====
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==== Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not. ====
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Considering this system, target proteins near the R9 peptides are taken by endocytosis.  
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Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis.  
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In other words, it can be said that it induces endocytosis regardless of whichever R9 peptides and target proteins are fused.
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So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.
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We tried to wonder whether we should fuse R9 and target proteins. <br>
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However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins. <br>
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From this system, we thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we considered that fusing R9 peptide with a target protein may leads to higher efficiency.'''
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We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. '''Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.'''
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'''So, we tried to fuse R9 peptides with target proteins to increase penetration efficiency'''
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'''So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency'''
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We transformed ''E.coli'', arranging R9's sequence and target protein's gene in line. And after that we tried to make ''E.coli'' express R9::GFP fusion protein.
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We transformed ''E.coli'', fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make ''E.coli'' express R9::GFP fusion protein.
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In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was poor in growth(Fig3-3). Then, we checked R9's effect on R9::GFP fusion protein's expression by Western blotting and RT-PCR. We succeeded in confirming the existence of the mRNA (Fig3-4), but we did not find the protein (Fig3-5). These results indicated poor translation or quick breakdown of the protein.  
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In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, ''E.coli'' expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein.  
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].  
We used the existing GFP generator part, [http://partsregistry.org/Part:BBa_I746915 BBa_I746915].  
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[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]
[[File:R9とGFPでやるやつさいしn.png|thumb|left|320px|Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from ''Arabidopsis thaliana'' 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide]]
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We wondered whether R9 peptides work properly and put FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, need to verify the function of R9 peptides. For this reason, we performed following experiment(Fig3-6).  
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It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6).  
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First, we removed the cuticle of ''Arabidopsis thaliana'''s leaves by scratching them. Second, we soaked the leaves into a solution of only GFP, or GFP and R9 for each. After 5 minutes we washed cells by PBS in order to put GFP and R9 peptides away from leaves. After that, we compared leaves having soaked in only GFP and ones having soaked R9 and GFP. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).
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First, we removed the cuticles from ''Arabidopsis thaliana'''s leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).
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The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or around plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.
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The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.
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==4. Activation==
==4. Activation==
[[File:アイコン4.png|250px|left|link=|Activation]]
[[File:アイコン4.png|250px|left|link=|Activation]]
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<br>On the final step; '''Activation'''. We verified whether FT protein made in'' E.coli'' worked normally in plant cells.<br><br>  
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<br>The final step is; '''Activation'''. We verified whether FT protein made by '' E.coli'' work normally in plant cells.<br><br>  
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Actually, FT protein is a transcriptional factor so that we can check the ability of FT protein by observing the transcript levels of genes FT protein upregulate. <br><br><br>
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Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT protein. <br><br><br>
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===The detail function of FT===
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FT protein increases transcript activities of flowering factors which lead to flower formation in a plant cell. 
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Let us introduce the function mechanism of FT protein. Actually, FT function is to up-regulate genes relating to bloom flowers. at the stage of flowering of plants, the expressions are incresed in these genes, FUL, SEP3, and so on. These genes work on shoot apex and change the form of shoot apex in order to start flowering. We focused on the amount of FUL and SEP3 because they are the most popular genes among them.
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===The detail function of FT as a transcriptional factor===
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[[File:Flowering factors1.png|thumb|center|610px|Fig.4-1 FT protein triggers some flowering genes and they induce flower formation. FT protein up-regulates some flowering genes and they induce plants to bloom. ]]
-
[[File:FULSPE3AP1.jpg|thumb|right|300px|Fig.4-1 FT protein triggers some flowering genes and they induce plants to bloom. FT protein up-regulates some flowering genes and they induce plants to bloom. ]]
+
-
FT protein increases transcript activity of several genes which leads to flower formation. The protein can . <br>
 
<br>
<br>
<br>
<br>
-
 
-
<br><br><br><br><br><br>
 
===Injecting FT and verifying its function by RT-PCR===
===Injecting FT and verifying its function by RT-PCR===
-
To evaluate the function of FT which our Fairy made, we used leaf cells of ''Arabidopsis thaliana '' , not soot apex. Plants produce FT protein in their leaves and carry it to soot apex. For these reason, we assumed that our Flower Fairy can make flowers bloom only if it can introduce active FT protein in leaves. In addition to that, we can get lots of sample easily. Previous research said that FT gene can also up-regulate some gene,such as FUL and SEP3, in leaves so that We injected FT protein into leaves and conducted RT-PCR to value the amount of expressed RNA.<br>
+
To evaluate the function of FT produced by our ''E.coli'', we tried to check the amount of flowering factors' mRNA upregulated by FT. Experimental procedure is as follows.<br>
-
FT protein is injected by a syringe. Two types of samples were prepared, one is treated with FT and the other is treated with GFP as a control. GFP was used as a control to confirm the result of RT-PCR was derived from FT protein, not protein injection itself. GFP is suitable for control experiments because  GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)
+
1. We prepared solutions. One is composed of R9 peptides and GFP, and the other is composed of R9 peptides and FT. GFP protein is used as a control group. 2. We injected these solutions into each leaf by a syringe. 3.After incubation, we tried to perform RT-PCR in order to check up-regulation of flowering factors.<br>  
-
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves. ]]
+
-
<br>We performed RT-PCR to compare mRNA expression of ''Arabidopsis'' leaves treated with/without FT.<br>
+
 +
We used leaf cells of ''Arabidopsis thaliana'', insted of cells of a shoot apex. This is because shoot apex' cells are too small to observe. FT protein upregulates FUL and SEP3 genes in leaf cells, too. GFP is suitable for control experiments because  GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)
-
Fig.4-2 is the result of RT-PCR.  
+
 
 +
 
 +
[[File:お注射 Kyoto.png|thumb|center|610px|Fig.4-2 injecting FT protein into plant leaves in order to verify the function of FT protein. ]]
 +
<br>
 +
[[File:RT-PCR6.jpeg|thumb|left|300px|Fig.4-2 6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br>
[[File:RT-PCR6.jpeg|thumb|left|300px|Fig.4-2 6 His tag fusion FT protein was purified with Ni-NTA agarose column.<br>
30mg  of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.
30mg  of leaves of ''Arabidopsis thaliana'' before bolting were used for one sample.
Line 248: Line 250:
Lane6: SEP3 (FT-R9 treated) <br>
Lane6: SEP3 (FT-R9 treated) <br>
Lane7: AP1 (GFP-R9 treated) amplicon 958bp<br>]]
Lane7: AP1 (GFP-R9 treated) amplicon 958bp<br>]]
-
[[File:TotalRNA20120925-01.png|thumb|300px|Fig.4-3 Electrophoresis of RNA.
+
[[File:jyoge.png|thumb|300px|Fig.4-3 Electrophoresis of RNA.
RNA concentration is adjusted.<br>
RNA concentration is adjusted.<br>
Lane1: RNA of 16h incubated leaves(GFP-R9 treated)<br>
Lane1: RNA of 16h incubated leaves(GFP-R9 treated)<br>
Lane2: RNA of 16h incubated leaves(FT-R9 treated)<br>
Lane2: RNA of 16h incubated leaves(FT-R9 treated)<br>
-
Lane3: RNA of fresh leaves]]
+
Lane3: RNA of fresh leaves]]<br clear="both" />
-
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
+
-
As shown above, we got no correct ampicon band, even of ''tubulin'', which is high expressed gene we used as internal control. One possible reason of this failure is the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.
+
As shown above, we got no correct ampicon band, even from ''tubulin'', which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.
-
As shown in the Fig.4-3, RNA samples in this time are degradated.<br>
+
As shown in the Fig.4-3, this time, RNA samples were degradated.<br>
-
Moreover, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br>
+
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)<br>
===Establishing an effective way of RNA purification===
===Establishing an effective way of RNA purification===
-
We found that RNA purification method should be improved for performing RT-PCR successfully .<br>
+
We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .<br>
We improved following things;<br>
We improved following things;<br>
1. Total leaf volume was increased.<br>
1. Total leaf volume was increased.<br>
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br>
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.<br>
3. We centrifuged samples and collected supernatant twice after adding ISOGEN.<br>
3. We centrifuged samples and collected supernatant twice after adding ISOGEN.<br>
-
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4, 4-5.) <br>
+
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4A.) <br>
Then we used better quality of RNA for reverse transcription and retried RT-PCR.
Then we used better quality of RNA for reverse transcription and retried RT-PCR.
-
Fig.4-6 shows the result of this RT-PCR.
+
Fig.4-4C shows the result of this RT-PCR.
<br><br>
<br><br>
-
[[File:RNA0925.png|thumb|300px|left|Fig.4-4 RNA waveform of long time incubated leaves]]
+
[[File:ABC.png|thumb|600px|center|Fig.4-4<br/>A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low<br/>
-
 
+
B: Improved RNA waveform<br/>
-
[[File:RNA0926.png|thumb|320px|Fig.4-5 Improved RNA waveform]]
+
C: Electrophoresis of RNA. RNA concentration is adjusted. These band showed all sample were purified correctly
 +
D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1]]
<br clear="both" />
<br clear="both" />
 +
===Conducting RT-PCR and qPCR again by using new RNA purification===
 +
[[File:RT-PCR_1026kyoto.png|thumb|300px|right|fig4-5 The result of RT-PCR<br>Lane1: TUBULIN (GFP-R9 treated) amplicon 61bp
 +
<br>Lane2: TUBULIN (FT-R9 treated)
 +
<br>Lane3: FUL (GFP-R9 treated) amplicon 132bp
 +
<br>Lane4: FUL (FT-R9 treated)
 +
<br>Lane5: SEP3 (GFP-R9 treated) amplicon 87bp
 +
<br>Lane6: SEP3 (FT-R9 treated)
 +
<br>Lane7: AP1 (GFP-R9 treated) amplicon 958bp]]After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.
 +
To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control.
 +
<br><br><br><br><br><br><br>
 +
===Checking FT protein purification by Western Blotting===
-
 
+
We could not confirm FT function though enough quality of RNA was extracted. Therefore, we questioned about FT protein quality, and performed western blotting to confirm FT protein is successfully purified.<br>
-
[[File:TotalRNA20120926.jpeg|thumb|300px|left|Fig.4-6]]
+
Unfortunately, the band of FT was not detected. Because of the time shortage, we could not conduct further experiment. We have to reconsider about FT purification and now we are trying.<br>
-
[[File:RT-PCR7.jpeg|thumb|300px|Fig.4-7 <br>
+
-
Lane1: 100bp ladder<br>
+
-
Lane2: TUBULIN <br>
+
-
Lane3: FUL<br>
+
-
Lane4: SEP3<br>
+
-
Lane5: AP1]]
+
-
<br clear="both" />
+
-
<br>
+
-
 
+
-
===Conducting RT-PCR and qPCR again by using new RNA purification===
+
-
After we purified RT-PCR via the new method, we performed RT-PCR again about SEP3 and FUL
+
-
 
+
-
===Checking whether FT protein really introduced or not===
+
==Achievement==
==Achievement==
Line 300: Line 300:
2.Secretion<br>
2.Secretion<br>
*Modify TorA signal to be easy to use the signal more
*Modify TorA signal to be easy to use the signal more
-
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABCD, pspA, double terminator
+
*Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABC, pspA, double terminator
*Standardize kil gene
*Standardize kil gene
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''
**Multiply TatABC in order to strengthen Tat secretion system--'''not yet'''
Line 314: Line 314:
=[[File:Kyoto_DiscussionHeader.png]]=
=[[File:Kyoto_DiscussionHeader.png]]=
-
In order to see whether FT is usable or not, we measured the amount of FUL and SEP3 by qPCR of cDNA. However, there was not any significant difference between experimental group and negative control.
+
At '''PENETRATION''' step, GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed.
-
It’s cause may be that FT protein was not active or decomposed in the cell or even that FT protein didn’t enter plant cells.
+
This is because there is an example where proteins tagged with arginine at C- terminals were correctly expressed whereas proteins tagged at N-terminals were not([http://partsregistry.org/wiki/index.php/Part:BBa_K249005 BBa_K249005] ).
 +
By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve the efficiency of macropinocytos, a type of endocytosis.
 +
 
 +
 
 +
At''' Activation''', in order to see whether FT is usable or not, we measured the relative RNA expression of FUL and SEP3 by qPCR. However, there was not any significant difference between experimental group and negative control.
 +
We found that FT was not successfully purified and there is the room for improvement in that.
 +
In addition of this, several reasons can be considered.
 +
The cause of failure may be that FT protein was not active or decomposed in the cell or even that FT protein didn't enter plant cells.
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.
If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.
Line 326: Line 333:
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed.  
If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed.  
-
When FT didn’t enter plant cell, we will reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.
+
When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s.
-
Wnen FT was decomposed in the cell, the problem may be solved by fusing R9 with FT’s C-terminal, not N-terminal, because there are some references mentioning that protein tagged with R9 at it’s N-terminal may be decomposed in the cell.
+
Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein.  
 +
 
 +
 
===Future Works===
===Future Works===

Latest revision as of 04:04, 27 October 2012

  • Kyoto Introduction.png
  • Kyoto Experiment.png
  • Kyoto Discussion.png
  • Kyoto Reference.png

Contents

Introduction

Realizing Flower Fairy in real world

FFT kyoto.png

Have you ever seen flower fairies? Probably the answer is "NO", (though some of you might have come across them in your childhood,) because they are imaginary creatures which exist only in fairy tales. Don’t you think it would be wonderful if you could live with flower fairies? In addition, their lovely power to make flowers bloom would be profitable for us in many ways, such as application to agriculture. This is why we have set our project for realizing Flower Fairy E.coli with synthetic biology!! In order to realize our dream, we focused on FT protein, known as Florigen.This protein is a kind of plant hormones. First of all, FT protein is produced in leaves, and then move to the shoot apex and bloom flowers. Therefore, FT protein is the key to our project.

Our Goal is to induce flower formation just by putting Flower Fairy E.coli on leaves!

When you want to use our Flower Fairy E.coli, all you have to do is just put them on plant leaves! When you spread Flower Fairy E.coli, FT protein is secreted and penetrate the cell membrane of a plant, and the plant starts blooming.

We had to go through four steps in order to achieve our goal――Flower Fairy E.coli.
These four steps are EXPRESSION,SECRETION, PENETRATION, and ACTIVATION

Kyoto wiki intro 1 Expression.png Kyoto wiki intro 1 Activation.png
Kyoto wiki intro 1 Secretion.png Kyoto wiki intro 1 Penetration.png


In each step, we have some problems to be attacked.
EXPRESSION; It is unclear whether E.coli can express FT protein, because FT protein is derived from plant cells .
SECRETION; After produced, ''E.coli'' have to secrete FT protein outside of themselves.
PENETRATION; FT protein has to penetrate into plant cells.
ACTIVATION; Even if FT protein could get into the cells, it is unclear whether FT protein produced by E.coli can activate genes in shoot apex cells and induce flower formation.

Kyoto Experiments&ResultsHeader.png

1.EXPRESSION

Expression

The first step is EXPRESSION.
We had to make E.coli produce FT protein.

However, in the natural environment, wildE.coli don't have FT gene. Therefore we tried to make a new BioBrick part including FT gene and introduce it into E.coli.






Modifying FT gene for Biobrick

FT gene is derived from Arabidopsis thaliana, a model plant. Professor Araki in Kyoto University kindly gave us FT cDNA in TOPO blunt end 2(Invitrogen). FT cDNA has two cleavage sites of iGEM restriction enzymes, EcoR1 and Pst1 (Fig.1-1 A), therefore we modified FT gene sequence by Inverse PCR with primers containing two base mismatches between primer and cDNA(Fig.1-1 B). As a result, we could get mutated FT cDNA, which are not cleaved by iGEM restriction enzymes, and then we added prefix and suffix to FT. Finally, we could make new BioBrick parts of FT (Fig.1-1 C).

Fig.1-1
A : Necessity for mutation of FT cDNA. FT sequence had two cleavage sites of iGEM restriction enzymes.
B : Inverse PCR Method
Inverse PCR is a measure of mutating FT gene segment. Inverse direction of primers and designing those including mismatch residue leads to mutation of the original plasmid.
C : Standardization of FT cDNA. We had mutated and added prefix and suffix to FT.


Confirming expression of FT

We constructed the plasmid shown in the Fig.1-2. One was FT gene with T7 promoter (BBa_I719005) and 6His tag, and the other was FT gene only with T7 promoter. T7 promoter is a strong promotor regulated by T7 polymerase. In our strain of E.coli, the expression of T7 polymerase can be induced by IPTG. 6His tag, which is used in later steps, enabled us to purify FT protein from E.coli using affinity chromatography.

We needed to confirm that E. coli can express FT protein, because there was a possibility that E.coli can not translate FT or FT is unstable or toxic in E.coli.
In order to confirm the expression of FT protein, we performed Western blotting using anti-FT antibody.
As a result, we observed FT and 6 His:FT bands at the expected molecular weight region(Fig.1-2).
So we successfuly confirmed the FT expression!

Fig.1-2 Construction of FT generator and the results of Western blotting against FT and 6 His:FT protein
Expression of T7 polymerase is regulated by IPTG. In both of constructions, left lane shows the band NOT induced by IPTG and right lane shows the band induced by IPTG.


We succeeded in confirming the mutation and the expression of FT and 6 His:FT protein in E.coli!

2.SECRETION

Secretion

The second step is SECRETION. We want E.coli to secrete FT protein to outside of the cell.
Now our E.coli can produce FT protein, but a big issue still remains: how they can transport proteins to the outside of the cells? Many people might think cell lysis is the best way. But in our project lysis is not very good, because it would possibly cause all fairies' deaths. We hoped to see the continuous effect of Flower Fairy E.coli, not just temporary. So, we adopted a method other than lysis which enables E.coli to transport FT protein continuously.

Proteins are required to go through two membranes

Fig.2-1 FT protein have two hurdles in order to get out of the cell

E.coli have two membranes: inner membrane and outer membrane. To transport FT protein to outside of E.coli, FT protein has to pass through the two membranes. So we searched for secretion systems to make FT protein go through these membranes.


TorA signal enables proteins to go through the inner membrane via Tat pathway

Fig.2-2 FT protein with TorA signal go through the inner membrane to the periplasm.

Wild E.coli have many secretion systems. In order to enable proteins to go through inner membrane, we decided to use one of these systems, called Twin Arginate Translocation (Tat) pathway. Tat pathway is more suitable than other secretion systems for our project, because by Tat pathway E.coli secrete proteins into the periplasm keeping proteins’ conformation and function. The following is the mechanism of Tat pathway; a Tat transporter recognizes TorA signal, and proteins having a TorA signal at their N terminals only can get into the periplasm.

We improved usability of TorA signal BioBrick

We tried to combine TorA signal with FT protein. Searching previous iGEM projects, we actually found TorA signals as iGEM parts (such as BBa_K638402) submitted by other teams. These parts, however, have two big problems. One problem is that these parts do not have RBS. So iGEMers who use the parts have to spend additional processing time. The other problem is that stop codon appear between signal region and target coding sequence when these parts are combined with some other parts by standard or 3A assembly. In short, when iGEMers use these parts, TorA-fusion protein would not be expressed at all, or only TorA would be expressed.

To solve these problems, we produced new applicable TorA signal BBa_K797002 (Fig.2-2). Our part has two improvements. BBa_K797002 contains RBS and indels to prevent the emergence of stop codon between signal region and target cording sequence. We sequenced BBa_K797002 and confirmed that stop codon did not appear when we used it in Standard and 3A assembly. Using green fluorescent protein (GFP) as a target protein, we constructed plasmid of plac-RBS-TorA-GFP-DT and introduced it into E.coli. After that, we observed the TorA-GFP-fusion-expressing cells (Fig.2-3) suggesting that the TorA-GFP fusion was successfully expressed. This meant RBS in our TorA signal worked well and stop codon didn't appear after assembly.

Fig.2-3 Previous TorA signal BioBricks (such as BBa_K638402) and our modified TorA signal (BBa_K797002)
Fig.2-4 E.coli (left) sample stained with Hoechst and observed with 352nm wavelength. Hoechst can stain DNA. E.coli (right) sample having plac-TorA-GFP-DT and observed with 489nm wavelength.

Kil protein enables proteins to go through outer membrane

Fig.2-5 Kil protein makes holes on outer membrane.

With Tat secretion pathway, FT protein can be transported into the periplasm. Next, FT protein needs to move to the outside of E.coli. For secretion, we used kil protein, which is derived from λ phage. Kil protein makes holes in the outer membrane of E.coli. So in our project, we introduced kil gene into E.coli. But the function of outer membrane is essential for E.coli to survive. Overexpression of kil gene, therefore, causes cell death. For this reason, we must check whether kil gene is harmful or not under our condition.

Kil protein had no significant effect on E.coli's growth

We made the construction plac-RBS-kil-double terminator, whose backbone is pSB3C5. After culturing for 18hr at 37℃, we removed the supernatant, and diluted it to OD600=0.1. Then we resuspend it. And then, we added 0/0.001/0.01/0.1/1mM IPTG to each. While culturing again at 37℃, we measured OD600, which indicate the density of E.coli. The figure below shows the results. These results indicated that the expression of plac-RBS-kil-DT (pSB3C5) makes no effect on the survival of E.coli.

We established the new biobrick for the useful secretion system. When we apply these systems, torA signal and kil protein, to Flower Fairy E.coli, we can make E.coli secrete FT protein without cell lysis!!(Fig.2-7)

Fig.2-6 The result of evaluation of kil protein.
Vertical axis means the value of OD 600. And horizontal axis means incubation time. Time caused changes of cell growth after IPTG induction.
Fig.2-7 Overall vision of Tat secretion system and kil protein. We apply this system for Flower Fairy E.coli.
1.Tat transporters are piercing in the inner membrane. They recognize TorA signal, and transport into the periplasm only proteins that have TorA signal at their N terminals. 2. Kil protein makes holes in the outer membrane of E.coli and the proteins go through the outer membrane.


Improvement of secretion system

Now we can let E.coli to secrete FT protein to the outside by using Tat pathway and kil protein. But E.coli secrete only a small amount of FT protein when they use Tat transporters which they inherently have. To make E.coli secrete enough amount of FT protein, we needed to improve the efficiency of their secretion systems. To reach this goal, we used two genes. One is composed of TatA, TatB, and TatC, which composes Tat transporter. Another is phage-shock protein A (pspA), which wild E.coli have. When their inner membrane is damaged, pspA gene is expressed ,and pspA maintains membrane potential and H+ concentration gradient between the periplasm and cytoplasm. It is known that pspA promote trasport of proteins to the periplasm through the detail mechanisms are unknown. As the next step of secretion, by the extra induction of these genes, we tried to increase the amount of secresion. We constructed Tat secretion cassette with constitutive promoter (BBa_K797004).(Fig.2-8) This part includes TatA, B and C proteins coding region and pspA. By using this part, the amount of Tat transporter is and pspA can be increased . In short, we can make E.coli secrete more proteins with TorA.(Fig.2-9)



Kyoto 2012 suggests this new way of secretion, Tat pathway and kil protein, and provides iGEMers with these genes regulated by constitutive promoter. We checked the sequence of TatABC (BBa_K797000) and the sequence of pspA (BBa_K797001) individually, and then, we made Tat construction composed of constitutive promoter (BBa_J23107), TatABC (BBa_K797000), pspA (BBa_K797001) and double terminator (BBa_B0015). We performed electrophoresis of this cassette confirmed the length of our parts and sequenced them partially.


3.PENETRATION

Penetration

The third step is PENETRATION. In order to induce flower formation, FT protein from E.coli must enter into plant cells. This is because FT protein upregulates other proteins which lead to flower formation in plant cells.

However, normally proteins cannot penetrate a cell membrane of a plant. Therefore, we needed a method to send FT protein into plant cells. Thanks to the advice from Doctor Washida, we found a method for the penetration of cell membrane. In this method, we use polyarginines called R9 peptides.

R9 peptide enables FT protein penetrate membranes by endocytosis
Fig.3-1 Image of R9 peptide : R9 peptide consists of 9 arginine residues.

R9 peptide consists of nine arginine residues(Fig.3-1). It is known as a kind of CPP (Cell Penetrating Peptide). Arginine-rich peptides induce macropinocytos, a type of endocytosis. From this, we judged that R9 peptides were suitable for cell membrane penetration.

This is the mechanism of how R9 peptides work(Fig3-2).

Firstly, R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.
Secondly, the cell responds to this stimulus and starts to endocytose.
Finally, proteins around the endocytosing region of the cell are taken into the cell.



R9 peptides seem to work effectively whether or not R9 peptides are fused with target proteins. However, there are few examples about endocytosis of plants, so we needed to check the function of R9 when used on plant cells.

Fig.3-2 Mechanism of endocytosis by R9 peptide
1.R9 peptides adhere to a cell membrane of a plant because of their hydrophobic character.
2.The cell responds to this stimulus and starts to endocytose.
3.FT protein around an invaginating region of the cell are taken into the cell.


Given the R9 system, it induces endocytosis whether R9 peptides and target proteins are fused or not.

Considering this system, target proteins near the R9 peptides are taken into cells by endocytosis. So, we assumed that the system would induce endocytosis in both cases where R9 peptides and target proteins were fused and not fused.
However, as we wanted to introduce proteins as efficiently as possible, we deliberated whether we should fuse R9 and target proteins.
We thought the shorter the distance between R9 peptides and target proteins is, the more easily they are taken into a plant cell. Therefore, we expected that fusing R9 peptide and a target protein would lead to higher efficiency.

So, we tried to fuse R9 peptides and target proteins to increase penetration efficiency

We transformed E.coli, fusing R9 pepetides and target protein's gene in line. By doing this, we tried to make E.coli express R9::GFP fusion protein.

In order to visualize the function of R9, we tried to prepare R9::GFP fusion protein. However, E.coli expressing the R9::GFP fusion protein was not effectively cultivated.(Fig3-3). Then, we checked whether R9 peptides had a bad effect on the expression of R9::GFP fusion protein by Western blotting and RT-PCR. Although we succeeded in confirming the existence of the mRNA (Fig3-4), we did not find the proteins (Fig3-5). These results are probably caused by poor translation or quick breakdown of the protein.

We used the existing GFP generator part, BBa_I746915.


We used R9 and GFP separately and to check the R9 peptides function

Fig.3-6 Preparation for samples verifying R9 function 1.Cutting leaves from Arabidopsis thaliana 2.Removing cuticle by scratching 3.Soaking them with solutions. (Left side, control group):a solution of only GFP (Right side, experimental group):that of GFP and R9 peptide

It was a question whether R9 peptides work properly and introduce FT protein into plant cells. Although endocytosis is often observed in animal cells, there are few examples of plant cell’s endocytosis. We, therefore, needed to verify the function of R9 peptides. For this reason, we performed the following experiment(Fig3-6).


First, we removed the cuticles from Arabidopsis thaliana's leaves by scratching them. Second, we devided the leaves into two groups, and soaked one into a solution of only GFP, and the other into that of GFP and R9. After 5 minutes we washed cells by PBS in order to wash away GFP and R9 peptides from around the leaves. After that, we contrasted leaves having soaked into the only GFP solution with ones having soaked into the solution of R9 and GFP mixture. Then we succeeded in getting the figure of GFP fluorescence (Fig3-7).


The control groups on the left were soaked in only GFP, and the experimental group on the right were soaked in GFP and R9. These fluorescence indicated that R9 peptides properly kept GFP in or on the plant cells. This figure strongly suggests that R9 peptides work successfully and GFP penetrate cell membrane, because this was taken by a confocal microscopy and seen as cross sections.

Fig.3-7 Verification of the R9 peptide function with use of GFP.Cells of Arabidopsis thaliana leaves soaked in a solution for five minutes, and Hoechst dyeing. Left side samples are soaked in only GFP, the right side samples in GFP and R9 peptide. From top to bottom, nuclei by Hoechst , GFP fluorescence (low magnification), nuclei by Hoechst , GFP fluorescence (high) (With confocal microscope Fluoview FU10i OLYMPUS)

We can make FT protein penetrate cell membrane of plants by R9 peptide function!.

4. Activation

Activation


The final step is; Activation. We verified whether FT protein made by E.coli work normally in plant cells.

Actually, FT protein is a transcriptional factor and we can check the ability of FT protein by observing the transcription levels of genes up-regulated by FT protein.


The detail function of FT

FT protein increases transcript activities of flowering factors which lead to flower formation in a plant cell. Let us introduce the function mechanism of FT protein. Actually, FT function is to up-regulate genes relating to bloom flowers. at the stage of flowering of plants, the expressions are incresed in these genes, FUL, SEP3, and so on. These genes work on shoot apex and change the form of shoot apex in order to start flowering. We focused on the amount of FUL and SEP3 because they are the most popular genes among them.


Fig.4-1 FT protein triggers some flowering genes and they induce flower formation. FT protein up-regulates some flowering genes and they induce plants to bloom.



Injecting FT and verifying its function by RT-PCR

To evaluate the function of FT produced by our E.coli, we tried to check the amount of flowering factors' mRNA upregulated by FT. Experimental procedure is as follows.
1. We prepared solutions. One is composed of R9 peptides and GFP, and the other is composed of R9 peptides and FT. GFP protein is used as a control group. 2. We injected these solutions into each leaf by a syringe. 3.After incubation, we tried to perform RT-PCR in order to check up-regulation of flowering factors.

We used leaf cells of Arabidopsis thaliana, insted of cells of a shoot apex. This is because shoot apex' cells are too small to observe. FT protein upregulates FUL and SEP3 genes in leaf cells, too. GFP is suitable for control experiments because GFP's molecular weight(27kDa) is relatively similar to that of FT(20kDa.)


Fig.4-2 injecting FT protein into plant leaves in order to verify the function of FT protein.


Fig.4-2 6 His tag fusion FT protein was purified with Ni-NTA agarose column.
30mg of leaves of Arabidopsis thaliana before bolting were used for one sample. FT or GFP protein and R9 peptide were diluted in PBS (pH7.4), 50ug/L and 500ug/uL each. Leaves were soaked into FT-R9 or GFP-R9 solution for 5min. and incubated for 16hr. in PBS(pH7.4.) After incubation, leaves were freezed with liquid nitrogen and glinded immediately.
Total RNA was extracted by phenol-chloroform extraction. cDNA was synthesized by reverse transcription and used as templates of RT-PCR. TUBULIN was used for internal control of mRNA expression.
Lane1: TUBULIN (GFP-R9 treated) amplicon 61bp
Lane2: TUBULIN (FT-R9 treated)
Lane3: FUL (GFP-R9 treated) amplicon 132bp
Lane4: FUL (FT-R9 treated)
Lane5: SEP3 (GFP-R9 treated) amplicon 87bp
Lane6: SEP3 (FT-R9 treated)
Lane7: AP1 (GFP-R9 treated) amplicon 958bp
Fig.4-3 Electrophoresis of RNA. RNA concentration is adjusted.
Lane1: RNA of 16h incubated leaves(GFP-R9 treated)
Lane2: RNA of 16h incubated leaves(FT-R9 treated)
Lane3: RNA of fresh leaves

As shown above, we got no correct ampicon band, even from tubulin, which is a high expressed gene we used as internal control. One possible reason of this failure was the poor quality of extracted RNA in this experiment. To check this, we compared the total RNA by electrophoresis, shown in fig.4-3.

As shown in the Fig.4-3, this time, RNA samples were degradated.
To add to this, the waveforms of them had a law peak at 260nm(Fig.4-4.)

Establishing an effective way of RNA purification

We found that RNA purification method needed to be improved in ordrer to perform RT-PCR successfully .
We improved following things;
1. Total leaf volume was increased.
2. Samples ware freezed with liquid nitrogen and suspended in ISOGEN more rapidly.
3. We centrifuged samples and collected supernatant twice after adding ISOGEN.
After the improvement, 260nm peak became higher and the degradation is minimized(Fig.4-4A.)
Then we used better quality of RNA for reverse transcription and retried RT-PCR. Fig.4-4C shows the result of this RT-PCR.

Fig.4-4
A: the waveform of RNA purified in the previous way. it indicated that RNA purity is low
B: Improved RNA waveform
C: Electrophoresis of RNA. RNA concentration is adjusted. These band showed all sample were purified correctly D: Lane1: 100bp ladder Lane2: TUBULIN Lane3: FUL Lane4: SEP3 Lane5: AP1


Conducting RT-PCR and qPCR again by using new RNA purification

fig4-5 The result of RT-PCR
Lane1: TUBULIN (GFP-R9 treated) amplicon 61bp
Lane2: TUBULIN (FT-R9 treated)
Lane3: FUL (GFP-R9 treated) amplicon 132bp
Lane4: FUL (FT-R9 treated)
Lane5: SEP3 (GFP-R9 treated) amplicon 87bp
Lane6: SEP3 (FT-R9 treated)
Lane7: AP1 (GFP-R9 treated) amplicon 958bp
After we purified RT-PCR via the new method, we performed RT-PCR again for SEP3 and FUL. The result is shown in fig. We were able to get sufficiently purified RNA, and amplify each gene.

To confirm the exact differnece between experimental group(FT+) and negative control(FT-), we conducted qPCR and compared the relative RNA expression of each gene. However, there was not significant difference between experimental group and negative control.






Checking FT protein purification by Western Blotting

We could not confirm FT function though enough quality of RNA was extracted. Therefore, we questioned about FT protein quality, and performed western blotting to confirm FT protein is successfully purified.
Unfortunately, the band of FT was not detected. Because of the time shortage, we could not conduct further experiment. We have to reconsider about FT purification and now we are trying.

Achievement

1.Expression

  • Mutate FT sequence
  • Standardize FT as an iGEM part
  • Confirm expression of FT protein in E.coli

2.Secretion

  • Modify TorA signal to be easy to use the signal more
  • Construct Tat secretion cassette that contains Constitutive promoter, RBS, TatABC, pspA, double terminator
  • Standardize kil gene
    • Multiply TatABC in order to strengthen Tat secretion system--not yet

3.Penetration

  • Keep GFP in or around plant cells using R9 peptide
    • Introduce FT in plant cells using R9 peptide--not yet

4.Activation

  • Get high quality of RNA
  • Amplify genes successfully
    • Check the function of FT--not yet

Kyoto DiscussionHeader.png

At PENETRATION step, GFP::R9 fusion protein connected at GFP’s N-terminal was not expressed. However, if GFP is tagged with R9 at C-terminal, that fusion protein may be successfully expressed. This is because there is an example where proteins tagged with arginine at C- terminals were correctly expressed whereas proteins tagged at N-terminals were not(BBa_K249005 ). By using GFP::R9 fusion protein connected at GFP's C-terminal, we might improve the efficiency of macropinocytos, a type of endocytosis.


At Activation, in order to see whether FT is usable or not, we measured the relative RNA expression of FUL and SEP3 by qPCR. However, there was not any significant difference between experimental group and negative control. We found that FT was not successfully purified and there is the room for improvement in that. In addition of this, several reasons can be considered. The cause of failure may be that FT protein was not active or decomposed in the cell or even that FT protein didn't enter plant cells. If we perform Western blotting of FT in plant cell after putting FT into it with R9, we will be able to understand whether the problem was FT protein or R9 peptides.

If the cause was that our FT protein was not active though FT had entered the cell, FT will be confirmed. In that case, we have to consider the possibilities that FT needs Post-translational modification or Arabidopsis thaliana that we used was old.

When FT needs Post-translational modification, we have to do more research about difference of Post-translational modification between E.coli and Arabidopsis thaliana.

When Arabidopsis thaliana that we used was old, it might have originally expressed enough FT, so our FT might not be necessary to induce FUL and SEP3.

If the cause was that FT didn’t enter plant cell or FT was decomposed in the cell, FT will not be confirmed. When FT didn’t enter plant cell, we have to reconstruct experimental system, for example, by using other kinds of R9 since there exist more effective R9s. Wnen FT was decomposed in the cell, we have to investigate the mechanism of decomposing protein.


Future Works

We noticed only flowering and florigen in this time but there are many other plant hormones. We made translocation pathway from E.coli into plant cells, so we will be able to introduce plant hormones into plant cells if E.coli can make them. It means we can control plant growth in any stage through genetically engineered E.coli. In the future that is not so far, we will be able to meddle in plants' growth――germinating, elongation, flowering, and fructification. We human will finally accomplish a technology that control plants perfectly.

Moreover, R9 peptide functions not only plant cell. R9 peptide works on animal cell similarly. It means that we found a pathway into any kinds of cells. R9 peptide tag enables us to introduce proteins into any cells, so we will be able to control all living cells using this technology.

Biosafety

BioSafetyLevel1


We cooperated with KAIT-Japan and the mark on the left indicates Biosafety Level of our parts.


Kyoto ReferencesHeader.png

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