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HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

Abstract
What`s T3SS
Detailed information about T3SS and summary of our achievements on iGEM 2010
Injection assay using onion cells
Experiments using plant cells are easier to perform than with mammalian ones
Ready-to-inject backbone and Bsa I cloning site
Ready-to-inject backbone and Bsa I cloning site enables easy fusion of T3S signal and protein
GSK tag system
A neat injection assay using GSK tag, which can specifically detect successfully injected proteins
Bsa I cloning site, RFC submission
Detailed documentation of costructing a BioBrick cloning site a BioBrick!

T3SS

Introduction

Fig. 1^[1]

Type III Secretion System (T3SS) is a system of pathogenic gram-negative bacterium such as Salmonella, Yersinia and EPEC(entero pathogenic E. coli). Using this system bacteria can inject whole protein molecules through a syringe like organelle named Type 3 Secretion Apparatus (Fig. 1). The target of this system is a eukaryotic cell. Naturally it is used to inject Virulence effector proteins.

Structure of Type III secretion apparatus

Type III secretion apparatus have a syringe like structure (Fig. 1). It can be visible under electron microscope ^[1]. Its length is about 80 nm and the diameter of its needle channel is about 2 nm ^[2]. The length is about 1/10 and the diameter is about 1/400 of an E. coli cell’s minor axis.

How does it function?

Fig. 2^[1]

When the needle tip attaches to a target cell membrane, a translocator complex is assembled on the target cell membrane. Inside the bacterial cell, an effector protein which has a unique secretion signal domain on its N-terminal is recognized by the specific chaperone, and forms effector-chaperone complex. This complex is recognized by the T3-secretion-associated ATPase located at the base of the needle complex (Fig. 2 i). Then, the ATPase stripes the chaperone from the complex and unfolds the effector protein to pass through the channel of the needle complex (Fig. 2 ii). Finally, the translocated effector is refolded within the target cell to carry out its function (Fig. 2 iii)^[1].

Our achievements on iGEM 2010

Fig. 3

We made a T3SS test construct on pSB1T3 vector, and transformed it to the E. coli (K-12) carrying BAC vector "pBAC-SPI-2" that encodes a part of Salmonella enterica serovar Typhimurium LT2 genome, which would express secretion apparatus (Fig. 3).

Secretion signal

It was reported that N-terminus 191 amino acid residues of SlrP (one of the natural effector protein of SPI-2 encoded T3SS) function as a T3 secretion signal domain in Salmonella^[3].

NLS (Nuclear Localization Signal)

In addition to the signal peptide, we located triple NLS (Nuclear Localization Signal) repeats between the T3 secretion signal and GFP, so that the injected GFP would be localized to the nucleus in the target cell.

RFP reporter

The RFP reporter gene constitutively produces RFP as an internal control that cannot be injected into target cells. This RFP would also play a role to distinguish GFP located in target cells from GFP remaining in bacterial cells under the fluorescence microscope.

Arabinose promoter

This [Secretion signal-NLS-GFP] fusion gene is under control of inducible arabinose promoter, this E. coli produces GFP only in the presence of arabinose.

Methods of Infection Assay

Fig. 4

We transferred E. coli [SPI-2/ T3 Signal-GFP, RFP] overnight LB culture to the neutral pH Mg²⁺ minimal medium (MgM pH=7.2) and cultured for 4 hours with arabinose to accumulate sufficient amount of GFP fusion protein (Fig. 4 i). Then the bacteria were transferred to acidic pH Mg²⁺ minimal medium (MgM pH=5.0) and cultured for 4 hours with arabinose to assemble T3 secretion apparatus^[4] (Fig. 4 ii). After that, bacteria were washed three times to remove contaminants that may detrimental for target cells (Fig. 4 iii). Since T3SS encoded in SPI-2 is known to be function inside of the phagosome of target cells^[5], the bacteria were exposed to the cells (RK13) under acidic pH condition (pH=5.0), which is considered to be similar to the environment inside of the phagosome.

Fig. 5

Under the acidic pH condition after the neutral pH, T3 secretion apparatus should be assembled (Fig. 5 i). First, translocator proteins are secreted through T3SS but effector proteins are not, because regulatory complex blocks the effectors at the base of the needle (Fig. 5 ii). However once the translocator complex assembled a pore on the target cell membrane the T3SS can sense the neutral pH condition of the cytosol (Fig. 5 iii) and this pH elevation removes the regulatory complex from the base of the needle (Fig. 5 iv) to start secretion of effectors. After exposure, the cells were incubated at 37C in 5.0% CO₂ and observed by fluorescent microscope.

Result and Discussion

Fig. 6

Cells were observed under Confocal Laser Scanning Microscope (OLYMPUS FV-1000D) with blue (473 nm) or green (559 nm) excitation lights at 7.5 hours after exposure. In the presence of arabinose E. coli expresses GFP and RFP, so bacterial cells are yellow( (Fig. 6 A-4), while GFP injected into target eukaryotic cells are green. Comparing Fig. 6 A-1 and A-4, GFP is located in the cytosol of target eukaryotic cells.

GFP translocated into the target cell didn’t show localization in the nucleus even 7.5 hours after exposure. These observations suggested that NLS domain in the GFP fusion construct we designed did not function as expected. This year we focus on this result, we aim to redesign the fusion protein vector, and to characterize its performance.

References

Galán JE, Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type III secretion machines. Nature 444: 567-573 Review [http://www.ncbi.nlm.nih.gov/pubmed/17136086 PubMed]
Ghosh P (2004) Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev 68: 771-795 Review [http://www.ncbi.nlm.nih.gov/pubmed/15590783 PubMed]
Miao EA, Miller SI (2000) A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium. Proc Natl Acad Sci U S A 97: 7539-7544 [http://www.ncbi.nlm.nih.gov/pubmed/10861017 PubMed]
Rappl C, Deiwick J, Hensel M (2003) Acidic pH is required for the functional assembly of the type III secretion system encoded by Salmonella pathogenicity island 2. FEMS Microbiol Lett 226: 363-372 [http://www.ncbi.nlm.nih.gov/pubmed/14553934 PubMed]
Waterman SR, Holden DW (2003) Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell Microbiol 5: 501-511 Review [http://www.ncbi.nlm.nih.gov/pubmed/12864810 PubMed]

HokkaidoU_Japan iGEM 2010 on Wiki, Poster and Presentation.

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 =T3SS=
-[[Image:HokkaidoU_T3SS_Fig1.jpg|right|thumb|300px|Fig. 1<br>Type III secretion apparatus<sup>[[#References|[2]]]</sup>.]]
 ==Introduction==
-&nbsp;&nbsp;&nbsp;Type III Secretion System (T3SS) is a system of pathogenic gram-negative bacterium such as ''Salmonella'', ''Yersinia'' and EPEC(entero pathogenic ''E. coli''). Using this system bacteria can inject whole protein molecules through a syringe like organelle named Type 3 Secretion Apparatus (Fig. 1). The target of this system is a eukaryotic cell. Naturally it is used to inject Virulence effector proteins.
+[[Image:HokkaidoU_T3SS_Fig1.jpg|right|thumb|300px|Fig. 1<sup>[[#References|[1]]]</sup>]]
+Type III Secretion System (T3SS) is a system of pathogenic gram-negative bacterium such as ''Salmonella'', ''Yersinia'' and EPEC(entero pathogenic ''E. coli''). Using this system bacteria can inject whole protein molecules through a syringe like organelle named Type 3 Secretion Apparatus (Fig. 1). The target of this system is a eukaryotic cell. Naturally it is used to inject Virulence effector proteins.
 ==Structure of Type III secretion apparatus==
-&nbsp;&nbsp;&nbsp;Type III secretion apparatus have a syringe like structure. It can be visible under electron microscope <sup>[[#References|[2]]]</sup>. Its length is about 80 nm and the diameter of its needle channel is about 2 nm <sup>[[#References|[3]]]</sup>. The length is about 1/10 and the diameter is about 1/400 of an ''E. coli'' cell’s minor axis. Thus this is the smallest injector in the world (Fig.1 a~c).
+Type III secretion apparatus have a syringe like structure (Fig. 1). It can be visible under electron microscope <sup>[[#References|[1]]]</sup>. Its length is about 80 nm and the diameter of its needle channel is about 2 nm <sup>[[#References|[2]]]</sup>. The length is about 1/10 and the diameter is about 1/400 of an ''E. coli'' cell’s minor axis.
 ==How does it function?==
-[[Image:HokkaidoU_T3SS_Fig2.jpg|right|thumb|300px|Fig.2<br>Signal peptide recognition and delivery<sup>[[#References|[2]]]</sup>.]]
+[[Image:HokkaidoU_T3SS_Fig2.jpg|right|thumb|300px|Fig. 2<sup>[[#References|[1]]]</sup>]]
+When the needle tip attaches to a target cell membrane, a translocator complex is assembled on the target cell membrane. Inside the bacterial cell, an effector protein which has a unique secretion signal domain on its N-terminal is recognized by the
-&nbsp;&nbsp;&nbsp;When the needle tip attaches to a target cell membrane, a translocator complex is assembled on the target cell membrane. Inside the bacterial cell, an effector protein which has a unique secretion signal domain on its N-terminal is recognized by the
+specific chaperone, and forms effector-chaperone complex. This complex is recognized by the T3-secretion-associated ATPase located at the base of the needle complex (Fig. 2 i). Then, the ATPase stripes the chaperone from the complex and unfolds the effector protein to pass through the channel of the needle complex (Fig. 2 ii). Finally, the translocated effector is refolded within the target cell to carry out its function (Fig. 2 iii)<sup>[[#References|[1]]]</sup>.
-specific chaperone, and forms effector-chaperone complex. This complex is recognized by the T3-secretion-associated ATPase located at the base of the needle complex (i). Then, the ATPase stripes the chaperone from the complex and unfolds the effector protein to pass through the channel of the needle complex (ii). Finally, the translocated effector is refolded within the target cell to carry out its function (iii)<sup>[2]</sup>.
 <div style="clear:both;"></div>
 =Our achievements on iGEM 2010=
-[[Image:HokkaidoU_T3SS_Fig3.jpg|300px|thumb|left|Fig.3<br>A model of GFP injection assay]]
+[[Image:HokkaidoU_T3SS_Fig3.jpg|300px|thumb|left|Fig. 3]]
-&nbsp;&nbsp;&nbsp;We made a T3SS test construct on pSB1T3 vector, and transformed it to the ''E. coli'' (K-12) carrying BAC vector "pBAC-SPI-2" that encodes a part of ''Salmonella enterica'' serovar Typhimurium LT2 genome, which would express secretion apparatus.
+We made a T3SS test construct on pSB1T3 vector, and transformed it to the ''E. coli'' (K-12) carrying BAC vector "pBAC-SPI-2" that encodes a part of ''Salmonella enterica'' serovar Typhimurium LT2 genome, which would express secretion apparatus (Fig. 3).
 ===Secretion signal===
-&nbsp;&nbsp;&nbsp;It was reported that N-terminus 191 amino acid residues of SlrP (one of the natural effector protein of SPI-2 encoded T3SS) function as a T3 secretion signal domain in Salmonella<sup>[[#References|[6]]]</sup>.
+It was reported that N-terminus 191 amino acid residues of SlrP (one of the natural effector protein of SPI-2 encoded T3SS) function as a T3 secretion signal domain in ''Salmonella''<sup>[[#References|[3]]]</sup>.
 ===NLS (Nuclear Localization Signal)===
-&nbsp;&nbsp;&nbsp;In addition to the signal peptide, we located triple NLS (Nuclear Localization Signal) repeats between the T3 secretion signal and GFP so that the injected GFP would localize inside of the nucleus in the target cell.
+In addition to the signal peptide, we located triple NLS (Nuclear Localization Signal) repeats between the T3 secretion signal and GFP, so that the injected GFP would be localized to the nucleus in the target cell.
 ===RFP reporter===
-&nbsp;&nbsp;&nbsp;The RFP reporter gene constitutively produces RFP as an internal control that cannot be injected into target cells. This RFP would also play a role to distinguish GFP located in target cells from GFP remaining in bacterial cells under the fluorescence　microscope.
+The RFP reporter gene constitutively produces RFP as an internal control that cannot be injected into target cells. This RFP would also play a role to distinguish GFP located in target cells from GFP remaining in bacterial cells under the fluorescence microscope.
 ===Arabinose promoter===
-&nbsp;&nbsp;&nbsp;This [Secretion signal-NLS-GFP] fusion gene is under control of inducible arabinose promoter, this ''E. coli'' produces GFP only in the presence of arabinose.
+This [Secretion signal-NLS-GFP] fusion gene is under control of inducible arabinose promoter, this ''E. coli'' produces GFP only in the presence of arabinose.
 <div style="clear:both;"></div>
-===Bacteria and cell culture condition===
-&nbsp;&nbsp;&nbsp;To perform the injection assay, we used LB medium (1.0% Bacto-Tryptone,  0.5% Bacto-yeast extract, 1.0% NaCl) and magnesium minimal medium (MgM) <sup>[[#References|[11]]]</sup> containing 170 mM MES-NaOH buffer(pH=5.0 or 7.2), 7.5 mM (NH<sub>4</sub>)2SO<sub>4</sub>, 5 mM KCl, 1 mM KH<sub>2</sub>PO<sub>4</sub>, 8 uM MgCl<sub>2</sub>, 38 mM glycerol and 0.1% casamino acids. We named these medium as MgM5(pH=5.0) and MgM7(pH=7.2). Also we used the acidic cell culture medium RPMI-10% FCS + HCl (pH 5.0) [RPMI5] and normal RPMI-10% FCS [RPMI7]. Bacteria were cultured at 37C with aeration and RK13 cells were cultured at 37C in 5.0% CO<sub>2</sub>. Appropriate antibiotics were added according to the resistance marker on each plasmid (25 ug/mL of chloramphenicol and 15 ug/mL of tetracycline). To induce GFP fusion protein L-arabinose was added to the medium at each step (final concentration = 0.4% ).
-===Methods of Injection Assay===
+==Methods of Infection Assay==
-[[Image:HokkaidoU_Japan_Fig6.jpg|200px|right|thumb|Fig.6<br>Model for control of effector translocation by the SPI-2 T3SS. (i) Following uptake into host cells, acidification of the vacuole lumen induces assembly of 9 the  secretion apparatus.  (ii) Membrane-associated SsaL/SsaM/SsaB (Fig.3)  regulatory complex  (in purple, black  and blue,  respectively) prevents premature  secretion of  effectors  (in brown). Translocon proteins (in green), connected to the T3SS apparatus, form a pore in the vacuolar membrane.  (iii) The pore enables a component(s) of  the T3SS  to sense  the elevated pH of the  host  cell  cytosol,  and  a  signal  is  transduced  to  the  SsaL/SsaM/SsaB  complex, which dissociates. (iv) Relief of effector secretion suppression enables their translocation <sup>[[#References|[10]]]</sup>.]]
+[[Image:HokkaidoU_T3SS_Fig4.jpg|right|thumb|300px|Fig. 4]]
+We transferred ''E. coli'' [SPI-2/ T3 Signal-GFP, RFP] overnight LB culture to the neutral pH Mg<sup>2+</sup> minimal medium (MgM pH=7.2) and cultured for 4 hours with arabinose to accumulate sufficient amount of GFP fusion protein (Fig. 4 i). Then the bacteria were transferred to acidic pH Mg<sup>2+</sup> minimal medium (MgM pH=5.0) and cultured for 4 hours with arabinose to assemble T3 secretion apparatus<sup>[[#References|[4]]]</sup> (Fig. 4 ii). After that, bacteria were washed three times to remove contaminants that may detrimental for target cells (Fig. 4 iii). Since T3SS encoded in SPI-2 is known to be function inside of the phagosome of target cells<sup>[[#References|[5]]]</sup>, the bacteria were exposed to the cells (RK13) under acidic pH condition (pH=5.0), which is considered to be similar to the environment inside of the phagosome.
-&nbsp;&nbsp;&nbsp;As mentioned before the T3SS encoded in SPI-2 naturally function inside of the phagosome of the target cell <sup>[[#References|[8]]]</sup>. So, it requires acidic pH to be assembled functionally <sup>[[#References|[7]]]</sup>. However, if the T3 apparatus is assembled successfully under low pH(pH=5.0) condition, only the translocator proteins are secreted through T3SS but effector proteins are not. And it was reported in 2010 that the translocator complex assembles a pore on the phagosome membrane of the host cell enabling the T3SS to sense the neutral pH condition of the cytosol, and this pH elevation switches the function of the T3SS to start secretion of effectors (Fig.6) <sup>[[#References|[10]]]</sup>. In addition we found that initial growth in MgM7 before the growth in MgM5 improve the production of GFP fusion protein in ''E. coli'' (data not shown). So, 10 hrs before exposure we transferred ''E. coli'' [SPI-2+GFP-T3signal+RFP] overnight culture from LB + arabinose to MgM7 + arabinose and grow for 4 hrs to charge sufficient amount of GFP fusion protein. 5.5 hrs before exposure, bacteria were transferred to MgM5 + arabinose and grow for 4 hrs to assemble T3 secretion apparatus. 1 hr before exposure, bacteria were washed with RPMI5 three times to remove toxin secreted from ''E. coli''. Then it was resuspended diluted with RPMI5 + arabinmose (final ΔOD = 0.06 at 600 nm). On the other hand RK13 cells were seeded on 6-well plate (2x 10<sup5</sup> cells/well) in antibiotics free RPMI7 at 20 hrs before exposure to the ''E. coli''. When the preparation is completed cell culture medium was replaced with 1 mL of the ''E. coli'' suspension (ΔOD = 0.06 at 600 nm) and incubate at 37C in 5.0% CO<sub>2</sub> to mimic the environment inside of the phagosome. At the same time samples of  arabinose(-) ''E. coli''[SPI-2+GFP-T3signal+RFP] and ''E. coli''[SPI-2 only] were prepared for the control condition.
-</div>
-<div style="clear:both"></div>
-=References=
-# Chen LM, Briones G, Donis RO, Galán JE. 2006. Optimization of the delivery of heterologous proteins by the Salmonella enterica serovar Typhimurium type III secretion system for vaccine development. Infect Immun. Vol.74:5826-5833. [http://www.ncbi.nlm.nih.gov/pubmed/16988261 PubMed]
-# Galán JE, Wolf-Watz H. 2006. Protein delivery into eukaryotic cells by type III secretion machines. Nature. Vol.444:567-573. Review. [http://www.ncbi.nlm.nih.gov/pubmed/17136086 PubMed]
-# Ghosh P. 2004. Process of protein transport by the type III secretion system. Microbiol Mol Biol Rev. Vol.68:771-795. Review.[http://www.ncbi.nlm.nih.gov/pubmed/15590783 PubMed]
-# Hansen-Wester I, Chakravortty D, Hensel M. 2004. Functional transfer of Salmonella pathogenicity island 2 to Salmonella bongori and Escherichia coli. Infect Immun. Vol.72:2879-2888. [http://www.ncbi.nlm.nih.gov/pubmed/15102800 PubMed]
-# Jacobi CA, Roggenkamp A, Rakin A, Zumbihl R, Leitritz L, Heesemann J. 1998. In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene. Mol Microbiol. Vol.30:865-882. [http://www.ncbi.nlm.nih.gov/pubmed/10094634 PubMed]
-# Miao EA, Miller SI. 2000. A conserved amino acid sequence directing intracellular type III secretion by Salmonella typhimurium. Proc Natl Acad Sci U S A. Vol.97:7539-7544. [http://www.ncbi.nlm.nih.gov/pubmed/10861017 PubMed]
-# Rappl C, Deiwick J, Hensel M. 2003. Acidic pH is required for the functional assembly of the type III secretion system encoded by Salmonella pathogenicity island 2. FEMS Microbiol Lett. Vol.226:363-372. [http://www.ncbi.nlm.nih.gov/pubmed/14553934 PubMed]
-# Waterman SR, Holden DW. 2003. Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell Microbiol. Vol.5:501-511. Review. [http://www.ncbi.nlm.nih.gov/pubmed/12864810 PubMed]
-# Wilson JW, Coleman C, Nickerson CA. 2007. Cloning and transfer of the Salmonella pathogenicity island 2 type III secretion system for studies of a range of gram-negative genera. Appl Environ Microbiol. Vol.73:5911-5918. [http://www.ncbi.nlm.nih.gov/pubmed/17675443 PubMed]
-# Yu XJ, McGourty K, Liu M, Unsworth KE, Holden DW. 2010. pH sensing by intracellular Salmonella induces effector translocation. Science. Vol.328:1040-1043. [http://www.ncbi.nlm.nih.gov/pubmed/20395475 PubMed]
-# Yu XJ, Liu M, Holden DW. 2004. SsaM and SpiC interact and regulate secretion of Salmonella pathogenicity island 2 type III secretion system effectors and translocators. Mol Microbiol. Vol.54:604-619. [http://www.ncbi.nlm.nih.gov/pubmed/15491354 PubMed]
+[[Image:HokkaidoU_T3SS_Fig5.jpg|left|thumb|500px|Fig. 5]]
+<div style="clear:both;"></div>
+Under the acidic pH condition after the neutral pH, T3 secretion apparatus should be assembled (Fig. 5 i). First, translocator proteins are secreted through T3SS but effector proteins are not, because regulatory complex blocks the effectors at the base of the needle (Fig. 5 ii). However once the translocator complex assembled a pore on the target cell membrane the T3SS can sense the neutral pH condition of the cytosol (Fig. 5 iii) and this pH elevation removes the regulatory complex from the base of the needle (Fig. 5 iv) to start secretion of effectors. After exposure, the cells were incubated at 37C in 5.0% CO<sub>2</sub> and observed by fluorescent microscope.
+<div style="clear:both;"></div>
+==Result and Discussion==
+[[Image:HokkaioU T3SS Fig6.png|right|thumb|400px|Fig. 6]]
+Cells were observed under Confocal Laser Scanning Microscope (OLYMPUS FV-1000D) with blue (473 nm) or green (559 nm) excitation lights at 7.5 hours after exposure. In the presence of arabinose ''E. coli'' expresses GFP and RFP, so bacterial cells are yellow(
+(Fig. 6 A-4), while GFP injected into target eukaryotic cells are green. Comparing Fig. 6 A-1 and A-4, GFP is located in the cytosol of target eukaryotic cells.
-{{:Team:HokkaidoU_Japan/footer}}
+GFP translocated into the target cell didn’t show localization in the nucleus even 7.5 hours after exposure. These observations suggested that NLS domain in the GFP fusion construct we designed did not function as expected. This year we focus on this result, we aim to redesign the fusion protein vector, and to characterize its performance.
+<div style="clear:both;"></div>
+=References=
+# Galán JE, Wolf-Watz H (2006) Protein delivery into eukaryotic cells by type III secretion machines. ''Nature'' 444: 567-573 Review [http://www.ncbi.nlm.nih.gov/pubmed/17136086 PubMed]
+# Ghosh P (2004) Process of protein transport by the type III secretion system. ''Microbiol Mol Biol Rev'' 68: 771-795 Review [http://www.ncbi.nlm.nih.gov/pubmed/15590783 PubMed]
+# Miao EA, Miller SI (2000) A conserved amino acid sequence directing intracellular type III secretion by ''Salmonella typhimurium''. ''Proc Natl Acad Sci U S A'' 97: 7539-7544 [http://www.ncbi.nlm.nih.gov/pubmed/10861017 PubMed]
+# Rappl C, Deiwick J, Hensel M (2003) Acidic pH is required for the functional assembly of the type III secretion system encoded by ''Salmonella'' pathogenicity island 2. ''FEMS Microbiol Lett'' 226: 363-372 [http://www.ncbi.nlm.nih.gov/pubmed/14553934 PubMed]
+# Waterman SR, Holden DW (2003) Functions and effectors of the ''Salmonella'' pathogenicity island 2 type III secretion system. ''Cell Microbiol'' 5: 501-511 Review [http://www.ncbi.nlm.nih.gov/pubmed/12864810 PubMed]
+HokkaidoU_Japan iGEM 2010 on [https://2010.igem.org/Team:HokkaidoU_Japan/Projects Wiki], [https://2010.igem.org/files/poster/HokkaidoU_Japan.pdf Poster] and [https://2010.igem.org/files/presentation/HokkaidoU_Japan.pdf Presentation].
+</div>
+{{Team:HokkaidoU_Japan/footer}}

Team:HokkaidoU Japan/Project/T3SS