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2024-03-28T16:12:23Z
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http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August27
Team:HokkaidoU Japan/Notebook/August27
2010-10-28T01:13:01Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August26|August 26]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August30|August 30]]</div></div><br />
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=Transfer [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] on pSB1A10 to pSB1C3=<br />
1.estimated concentrations of pSB1C3 and [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] which was amplified by miniprep before<br />
<br />
2.mixed solutions according to the table below<br />
<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center;" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
|pSB1C3<br />
|5 uL<br />
|-<br />
|DW<br />
|75 uL<br />
|-<br />
|10x M Buffer<br />
|10 uL<br />
|-<br />
|0.1%BSA<br />
|10 uL<br />
|-<br />
|EcoRI<br />
|5 uL<br />
|-<br />
|PstI<br />
|5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''100 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center;" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-1A<br />
|8 uL<br />
|-<br />
|DW<br />
|6 uL<br />
|-<br />
|10x M Buffer<br />
|2 uL<br />
|-<br />
|0.1%BSA<br />
|2 uL<br />
|-<br />
|EcoRI<br />
|1 uL<br />
|-<br />
|PstI<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div><br />
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3.incubated at 37C for 60min<br />
<br />
4.purified the two samples<br />
<br />
5.added 250 uL 100% EtOH to each samples<br />
<br />
6.centrifuged at 4C, 15000rpm for 10 min<br />
<br />
7.discarded supernatant<br />
<br />
8.put the samples into desiccator<br />
<br />
9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A<br />
<br />
10.mixed 1 uL pSB1C3 and 2 uL 1-1A<br />
<br />
11.added 3 uL ligation solution and 0.5 uL T4 ligase<br />
<br />
12.transfered the sample to a tube of 500 uL<br />
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13.incubated at 16C for 30 min<br />
<br />
->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.<br />
<br />
<br />
=Preparation of 3 Piece Ligation=<br />
<br />
*Parts Information<br />
<br />
{|border="1" style="margin-left: 20px;" class="protocol"<br />
|-<br />
!Name<br />
!Biobrick Name<br />
!Well<br />
!Length<br />
|-<br />
| Heat Sensor<br />
| BBa_K098995<br />
| 3-1E<br />
| 937 bp<br />
|-<br />
| RBS<br />
| BBa_B0034<br />
| 1-2M<br />
| 12 bp<br />
|-<br />
| RFP<br />
| BBa_E1010<br />
| 1-18F<br />
| 681 bp<br />
|-<br />
| Double Terminator<br />
| BBa_0015<br />
| 1-23L<br />
| 129 bp<br />
|}<br />
<br />
<br />
1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator<br />
<br />
2.mixed solutions according to the table below<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 13.5 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 0.1%BSA<br />
| 5 uL<br />
|-<br />
| EcoRI<br />
| 3 uL<br />
|-<br />
| SpeI<br />
| 1 uL<br />
|-<br />
| Heat Sensor<br />
| 22.5 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|} <br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 34.9 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 0.1%BSA<br />
| 5 uL<br />
|-<br />
| XbaI<br />
| 1 uL<br />
|-<br />
| PstI<br />
| 2 uL<br />
|-<br />
| RBS<br />
| 34.9 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}<br />
</div><br />
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<div style="float:left;"><br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 25.5 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 0.1%BSA<br />
| 5 uL<br />
|-<br />
| EcoRI<br />
| 3 uL<br />
|-<br />
| SpeI<br />
| 1 uL<br />
|-<br />
| RFP<br />
| 10.5 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 34 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 0.1%BSA<br />
| 5 uL<br />
|-<br />
| XbaI<br />
| 1 uL<br />
|-<br />
| PstI<br />
| 2 uL<br />
|-<br />
| Double Terminator<br />
| 3 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Projects/PCR
Team:HokkaidoU Japan/Projects/PCR
2010-10-28T00:56:31Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<br />
<br />
== PCR Based Assembly Protocol ==<br />
<br><br />
<br />
Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't by amplified. In moments like these PCR is handy. PCR is also great because it doesn't have many steps, the more steps the more ways to fail.<br />
<br />
<br><br />
<br />
When amplifying BioBricks primers which anneal to prefix and suffix are used. Good thing about them is that they are universal so with only 2 you can amplify all BioBricks. But unlike miniprep product BioBrick amplified like this don't produce 2 distinct bands when digested. So you are left wondering if digestion went well. <br />
<br />
<br><br />
<br />
We created a set of primers which solves this problem. You can judge if digestion was successful by presence of approximatively 100 bp or/and 200 bp bands and significant change in PCRed fragments length.<br />
<br />
<br><br />
<br />
So idea behind this primers quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the part like in Fig.1 bellow.<br />
<br />
<br><br />
<br />
[[Image:HokkaidoU_Japan_PCR_Protocol_Fig1.png|Fig.1]]<br />
<br />
<br><br />
[[Image:HokkaidoU_Japan_PCR_Protocol_Fig2.png|thumb|Fig.2]]<br />
Now when digested PCRed fragment produces distinct approx 100 bp or/and 200 bp fragment depending on cut sites. Knowing this you can continue to the next step with the sure and light heart (see Fig.2). This is crucial when you take into account that many participants have limited at the bench experience before hand.<br />
<br />
<br><br />
<br />
As everyone have guest by now these primers anneal to plasmid regions flanking the BioBrick. And there are more than 40 different primers. But not all of them are quite so different in fact all 7 registries assembly plasmids share the same sequence on the BioBrick flanking regions. And few others do also, bringing the number up to about 10. <br />
<br />
<br><br />
<br />
And with the new registry requirement to submit all BioBric in pSB1C3 plasmid present the possibility that in near future that many if not all plasmids could be supported by this primers making assembly easier.<br />
<br />
<br><br />
<br />
<br />
== Key Components of PCR Based Assembly Protocol ==<br />
<br />
*Reliable proofreading PCR enzymes like KOD Plus NEO<br />
**80 times more accurate than taq (At the moment we are not endorsed by producer : TOYOBO. Maybe in the future)<br />
*Digestion Visualization Primers (DV primers)<br />
*A program to calculate mixes for digestion and ligation<br />
<br />
<br />
<br />
== About the program to calculate mixes==<br />
<br />
<br />
We made simple program using excel to calculate restriction enzyme digestion and ligation mixes. One only needs to choose:<br />
<br />
*2 part to ligate<br />
*Primers used in PCR<br />
*DNA concentration after PCR<br />
*Part and plasmid ratio<br />
<br />
And amount for other reagents will be displayed automatically. This spares everyone from mundane calculations and help to conserve reagents. This program was made only for 2010 kit and currently isn’t available publically. But we feel the need for program like this to be on BioBrick registry page and could do calculations for all the parts in the registry. This is our proposal for improving efficiency of part assembly. Please contact us if you have any suggestions.<br />
<br />
[[Image:HokkaidoU_Japan_PCR_Protocol_Fig6.png|700px|Fig.3]]<br />
<br />
<br />
<br />
<br />
== Some other cool thing that you can do with these primers ==<br />
<br />
<br />
1)When you got small amount of plasmid DNA (like the Red DNA of the Kit) you can go from it to ligation the same day by PCR. And using these primers you can make sure that everything is going well. This good to know when you want to use a part you didn't plan for and you need it fast.<br />
<br />
<br><br />
<br />
2)When using PCR parts size also comes into question. Small pieces like RBS or NLS signal are so small that they go right through purification filters. Purification kit suggested recovery rate for these parts is as small as 26% (for 50bp). Although we ordered oligos for RBS we used and NLS signals was included inside primers we suggest that in theory it is possible to use visualization primers to adding more bulk to PCRed part thus increasing it’s recovery rate.<br />
<br />
[[Image:HokkaidoU_Japan_PCR_Protocol_Fig3.png|800px|Fig.4]]<br />
<br />
<br><br />
After electrophoresis gel extraction needs to be performed. But recovery rate form small parts like RBS negligible. By adding bulk you increase recovery rate for filters. And bands should show in gel<br />
<br><br />
<br />
[[Image:HokkaidoU_Japan_PCR_Protocol_Fig4.png|800px|Fig.5]]<br />
<br />
<br><br />
After recovery and ligation with the part of your choice you just cut the bulk.<br />
<br><br />
<br />
[[Image:HokkaidoU_Japan_PCR_Protocol_Fig5.png|800px|Fig.6]]</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Projects
Team:HokkaidoU Japan/Projects
2010-10-28T00:48:44Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<html><br />
<style><br />
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=Dr. ''E. coli'': The smallest injector in the world=<br />
==Introduction==<br />
&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. The target of this system is a eukaryotic cell. Naturally it is used to inject Virulence effector proteins.<br />
<br />
==Structure of Type III secretion apparatus==<br />
[[Image:HokkaidoU Japan Fig1.jpg|right|thumb|Fig.1<br>Needle complex of ''Salmonella typhimurium'' <sup>[[#References|[2]]]</sup>.]]<br />
<div class="narrow"><br />
&nbsp;&nbsp;&nbsp;Type III secretion apparatus have a syringe like structure. It can be visible under electro microscope <sup>[[#References|[2]]]</sup>. Its length is about 80nm and the diameter of its needle channel is about 20Å <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).<br />
</div><br />
<div style="clear:both"></div><br />
<br />
==How does it function?==<br />
[[Image:HokkaidoU_Japan_Fig2.jpg|right|thumb|Fig.2<br>Model for substrate recognition and delivery of proteins by type III secretion machines <sup>[[#References|[2]]]</sup>.]]<br />
<div class="narrow"><br />
&nbsp;&nbsp;&nbsp;When the needle tip attaches to the host cell membrane, a translocator complex that is also secreted by the T3SS is assembled on the host cell membrane and mediates the passage of the effector proteins through the target cell membrane. On the other hand an effector protein, which have a unique T3SS secretion signal domain on its N-terminal, is recognized by the specific chaperone and form an effector-chaperone complex. The secretion machinery, including a T3-secretion-associated ATPase, recognizes the complex. Then, the ATPase stripes the chaperone from the complex, which remains within the bacterial cell, and mediates the unfolding and threading of the effector protein through the central channel of the needle complex. Finally, the translocated effectors re-fold within the host cell to carry out their function (Fig.2).<br />
</div><br />
<div style="clear:both"></div><br />
<br />
==Motivation==<br />
&nbsp;&nbsp;&nbsp;It is valuable to develop a system that can modulate cell's behavior impersistently by injecting a desired "protein" directly into cells using a non-pathogenic strain. This system can be applied for many ways. For example, <br />
* Inject p53 to kill cancer cells selectively<br />
* Inject Yamanaka factors to induce iPS cells<br />
&nbsp;&nbsp;&nbsp;Fortunately, it was reported in 2007 that T3SS encoded in Salmonella Pathogenesity Island 2(SPI2) is functional in vitro on the ''E. coli'' (K-12) strain <sup>[[#References|[9]]]</sup>. However, the T3SS encoded in SPI-2 naturally function inside of the phagosome of the target cell <sup>[[#References|[8]]]</sup>. So, there was no report about whether the SPI-2 T3SS, that is cloned on ''E. coli'' (K-12) can inject a heterologous protein from outside of the target cell or not. That is why we have decided to put this challenging project into practice.<br />
<div style="clear:both"></div><br />
<br />
==Objectives of iGEM 2010==<br />
&nbsp;&nbsp;&nbsp;Though it is so exciting to create a cancer killer or an iPS generator ''E. coli'', it is too difficult to aim these practical goals from the very first; because it was reported that some domains are not able to pass the needle of T3SS <sup>[[#References|[1]]][[#References|[3]]]</sup>. So, we have established four objectives of iGEM 2010 as following.<br />
* To make T3SS available for ''E. coli''<br />
* To inject desired proteins into target eukaryotic cells<br />
* To determine whether the injected protein function correctly<br />
* To deliver the injected protein toward desired compartment in the target cell<br />
<div style="clear:both"></div><br />
<br />
==Detail information about SPI-2 region and SPI-2 carrying ''E. coli'' strain==<br />
[[Image:HokkaidoU_Japan_Fig3.jpg|200px|thumb|Fig.3<br>''S. enterica'' serovar Typhimurium SPI-2 region. A map of the ''S. enterica'' serovar Typhimurium SPI-2 region is depicted <sup>[[#References|[9]]]</sup>.]]<br />
[[Image:HokkaidoU_Japan_Fig4.jpg|200px|thumb|Fig.4]]<br />
<div class="narrow"><br />
&nbsp;&nbsp;&nbsp;Here we show the map of SPI-2 region (Fig.3). The length of SPI-2 region about 40kb and T3 secretion apparatus is a complex consisted of more than 20 kinds of proteins. As mentioned before cloned SPI-2 on R995 vector was functional in ''E. coli'' (K-12) <sup>[[#References|[9]]]</sup>. And also it was reported that SPI-2 region cloned on pBeloBAC11 (BAC=Bacteria Artificial Chromosome) is functional <sup>[[#References|[4]]]</sup>. So, we decided to request a ''E. coli'' (K-12) strain carrying a pBeloBAC11 vector encoding a genome fragment of ''Salmonella enterica'' serovar Typhimurium LT2 which covers the SPI-2 region [B_STM07H21 SGSC4024 1464540~1562427] from Salmonella Genetic Stock Center(SGSC) in University of Calgary, Canada (http://people.ucalgary.ca/~kesander/index.html). We confirmed the presence of the cloned SPI-2 region in this strain via PCR analysis(Fig.4) shall refer to it as "pBAC-SPI-2" from now on. Because of the provision of MTA, we cannot register this strain as a Bio Brick, but you can also get the same strain from SGSC.<br />
</div><div style="clear:both"></div><br />
<br />
==Construction of ''E. coli'' base GFP injector==<br />
[[Image:HokkaidoU_Japan_Fig5.jpg|200px|thumb|right|Fig.5]]<br />
<div class="narrow"><br />
&nbsp;&nbsp;&nbsp;We have made a T3SS test construct on the tetracycline resistant plasmid [pSB1T3] and transform it to the ''E. coli'' (K-12) carrying pBAC-SPI-2 as shown in (Fig.5). 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>. And it was also reported that GFP can be secreted through T3SS of ''Yersinia'' <sup>[[#References|[5]]]</sup>. So, we fused the N-terminus 191 a.a. of SlrP with the N-terminal of GFP. In addition 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. This [T3signal-NLS-GFP] fusion gene is under control of inducible arabinose promoter. In contrast RFP reporter gene, which is joined to the down stream of the GFP construct, is under control of a constitutive promoter. So, if arabinose is added to the culture medium, ''E. coli'' produces both GFP and RFP so that the bacteria become yellow. However, if arabinose is not added, only RFP is produced so that the bacteria become red. We have registered the T3SS signal domain mentioned above as a new BioBrick part.<br />
</div><div style="clear:both"></div><br />
<br />
==Bacteria and cell culture condition==<br />
&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% ).<br />
<br />
==Methods of Injection Assay==<br />
[[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>.]]<br />
<div class="narrow"><br />
&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.<br />
</div><br />
<div style="clear:both"></div><br />
<br />
==Results (7.5h after infection)==<br />
[[Image:HokkaidoU_Japan_Fig7.jpg|right|thumb|350px|Fig.7<br>[A-1]~[A-4] are the images of [S+G+R]. [B-1]~[B-4] are the images of [S+R]. [C-1]~[C-4] are the images of [S]. [X-1] is the phase contrast image. [X-2] is the green fluorescence image excited by blue laser. [X-3] is the red fluorescent image excited by green laser. And [X-4] is the merged image of [X-1]~[X-3]. ]]<br />
[[Image:HokkaidoU_Japan_Fig8.jpg|350px|right|thumb|Fig.8<br>wider angle merged images (A~C) are presented (A: [S+G+R], B: [S+R], C: [S]) and in (A’) only yellow color, which represents the ''E. coli'' cells, is deleted from (A).]]<br />
&nbsp;&nbsp;&nbsp;From now on we shall refer to "[SPI-2+T3signal-GPP+RFP]0.4% arabinose(+)" as [S+G+R], "[SPI-2+T3signal-GPP+RFP] arabinose(-)" as [S+R], and "[SPI-2 only]" as [S].<br />
We observed the cells by Cofocal Laser Scannning Microscope(OLYMPUS FV-1000D) under blue( nm) and green( nm) excitation light 7.5 hrs after the first exposure (Fig.7). [S+G+R] ''E. coli'' express both GFP and RFP, so in [A-4] ''E. coli'' cells appear to be yellow and GFP outside of ''E. coli'' appear to be green. Comparing [A-1] and[A-4] you can recognize that GFP is located in the cytosol of RK13. In contrast in [B-4] and [C-4] you cannot see GFP in the cytosol (Fig.8). In (A’) you can find that GFP is located in the shape of the cytosol more clearly. In fact GFP began to diffuse in the cytosol 4 hrs after the exposure (data not shown). However, GFP didn’t localize into the nucleus against our expectation even 7.5 hrs after exposure.<br />
<div style="clear:both"></div><br />
<br />
==Discussion==<br />
&nbsp;&nbsp;&nbsp;According to the results it can be suggested that SPI-2 encoded T3SS can inject a heterologous protein(GFP) from outside of the target cell even if it is cloned onto the heterologous host ''E. coli'' (K-12) and that injected GFP was functional in the target cell. In the process of our project, even if the ''E. coli'' is prepared under acidic pH condition, GFP was not injected into RK13 when the exposure step is performed under neutral pH condition (data not shown), suggesting that acidic pH is required for exposure step to use this system. By the way GFP didn’t localize into the nucleus against our expectation. So, NLS domain might not be able to function correctly in the GFP fusion protein constructed this time.<br />
<br />
==Possible Future Plans==<br />
* Collect quantitative and chronological data.<br />
* Clone the [T3signal-NLS-GFP] construct on a mammalian expressing vector to check the behavior of this fusion protein expressed inside of RK13.<br />
* Join NLS with the C-terminal of GFP.<br />
<br />
<br />
=References=<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]<br />
# 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]</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan
Team:HokkaidoU Japan
2010-10-28T00:42:13Z
<p>Sho N: </p>
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<br />
----<br />
=[[Team:HokkaidoU_Japan/Projects|Projects]]=<br />
<div id="projects" class="homepics">[[Team:HokkaidoU_Japan/Projects| ]]</div><br />
==[[Team:HokkaidoU_Japan/Projects|Dr. ''E. coli'' : The smallest protein injector in the world]]==<br />
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.<br />
<br />
==[[Team:HokkaidoU_Japan/Projects/PCR|PCR Based Assembly Protocol]]==<br />
&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Team|Team]]=<br />
<div id="team" class="homepics">[[Team:HokkaidoU_Japan/Team| ]]</div><br />
&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from faculty of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=<br />
<div id="notebook" class="homepics">[[Team:HokkaidoU_Japan/| ]]</div><br />
&nbsp;&nbsp;&nbsp;<br />
The record of our triumphs and failures. Well big part of it is failures. But we did it.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Protocols|Protocols]]=<br />
<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div><br />
&nbsp;&nbsp;&nbsp; <br />
General protocols we used.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Parts|Parts]]=<br />
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div><br />
&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.<br />
<br />
&nbsp;&nbsp;&nbsp;Also, we registerd one primer set which is useful for the PCR based protocol.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Safety|Safety]]=<br />
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div><br />
&nbsp;&nbsp;&nbsp;Addressing bio safety concerns<br />
<br />
----<br />
<br><br><br><br><br />
=Acknowledgements=<br />
==Relative to Labs==<br />
* Ken-ichi Yamazaki<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
*Hideaki Higashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Hisatoshi Shida<br />
** Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University<br />
* Takashi Oohashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Members of Yamazaki Lab<br />
*Masaaki Morikawa<br />
** Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
* Hidetoshi Okuyama<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
<br />
==Schools==<br />
* Salmonella Genetic Stock Center/University of Calgary<br />
* Graduate School of Environmental Earth Science<br />
* Department of biological science/School of Sciense<br />
* Institute for Genetic Medicine<br />
<br />
==Companies==<br />
* Amino Up Chemical<br />
* ECONIXE<br />
* Cosmo Bio<br />
* Mendel Workshop<br />
<br />
==Person==<br />
* Tadasu Emoto<br />
**Manager of ECONIXE<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/3/38/HokkaidoU_Japan_HomeTeam2.jpg"><br />
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</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan
Team:HokkaidoU Japan
2010-10-28T00:39:35Z
<p>Sho N: </p>
<hr />
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<br />
----<br />
=[[Team:HokkaidoU_Japan/Projects|Projects]]=<br />
<div id="projects" class="homepics">[[Team:HokkaidoU_Japan/Projects| ]]</div><br />
==[[Team:HokkaidoU_Japan/Projects|Dr. ''E. coli'' : The smallest protein injector in the world]]==<br />
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.<br />
<br />
==[[Team:HokkaidoU_Japan/Projects/PCR|PCR Based Assembly Protocol]]==<br />
&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Team|Team]]=<br />
<div id="team" class="homepics">[[Team:HokkaidoU_Japan/Team| ]]</div><br />
&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from faculty of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=<br />
<div id="notebook" class="homepics">[[Team:HokkaidoU_Japan/| ]]</div><br />
&nbsp;&nbsp;&nbsp;<br />
The record of our triumphs and failures. Well big part of it is failures. But we did it.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Protocols|Protocols]]=<br />
<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div><br />
&nbsp;&nbsp;&nbsp; <br />
General protocols we used.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Parts|Parts]]=<br />
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div><br />
&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.<br />
<br />
&nbsp;&nbsp;&nbsp;Also, we registerd one primer set which is useful for the PCR based protocol.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Safety|Safety]]=<br />
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div><br />
&nbsp;&nbsp;&nbsp;Addressing bio safety concerns<br />
<br />
----<br />
<br><br><br><br><br />
=Acknowledgements=<br />
==Relative to Labs==<br />
* Ken-ichi Yamazaki<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
*Hideaki Higashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Hisatoshi Shida<br />
** Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University<br />
* Takashi Oohashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Members of Yamazaki Lab<br />
*Masaaki Morikawa<br />
** Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
* Hidetoshi Okuyama<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
<br />
==Schools==<br />
* Salmonella Genetic Stock Center/University of Calgary<br />
* Graduate School of Environmental Earth Science<br />
* Department of biological science/School of Sciense<br />
* Institute for Genetic Medicine<br />
<br />
==Companies==<br />
* Amino Up Chemical<br />
* ECONIXE<br />
* Cosmo Bio<br />
* Mendel Workshop<br />
<br />
==Person==<br />
* Tadasu Emoto<br />
**Manager of ECONIXE<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/3/38/HokkaidoU_Japan_HomeTeam2.jpg"><br />
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</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Team
Team:HokkaidoU Japan/Team
2010-10-28T00:33:55Z
<p>Sho N: </p>
<hr />
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height:106px;<br />
float:left;<br />
margin:5px;<br />
}<br />
<br />
</style><br />
</html><br />
=Members=<br />
[[Image:HokkaidoU_Japan_logo.png|thumb|200px|right|HokkaidoU Japan Logo]]<br />
==Instructor==<br />
<div class="profile" style="width:520px;"><div id="yamazaki" class="profilepics"></div><br />
'''Ken-ichi Yamazaki'''<br />
* Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science<br />
* [http://noah.ees.hokudai.ac.jp/emb/ymzklab/yamazaki.html Yamazaki Lab]<br />
He helped every step of the way with his advice.<br />
</div><div style="clear:both"></div><br />
<br />
==Undergrad students==<br />
<div class="profile"><div id="vanagas" class="profilepics"></div><br />
'''Laurynas Vanagas'''<br />
* Department of Biological Sciences, School of Sciences<br />
* ''Team Leader''<br />
He has a late clock. But it's why he works hard midnight for team.<br />
<br />
</div><br />
<br />
<div class="profile"><div id="makino" class="profilepics"></div><br />
'''Shun-ichi Makino'''<br />
* School of Medicine<br />
* ''Project Leader''<br />
He got that crazy idea, the type 3 secretion system in E.coli.<br />
</div><br />
<br />
<div class="profile"><div id="niinuma" class="profilepics"></div><br />
'''Sho Niinuma'''<br />
* Department of Biological Sciences, School of Sciences<br />
The one who is first to come with the ideas why something was failed or why the results differ. <br />
</div><br />
<br />
<div class="profile"><div id="takahashi" class="profilepics"></div><br />
'''Kazuki Takahashi'''<br />
* Department of Biological Sciences, School of Sciences<br />
Takahashi keeps wiki in shape also responsible form team fitness and daily exercise.<br />
</div><br />
<br />
<div class="profile"><div id="matsumoto" class="profilepics"></div><br />
'''Yuichi Matsumoto'''<br />
* Department of Biological Sciences, School of Sciences<br />
In good ol' days he would go night fishing. And enjoy gourmet breakfast. But now it's night iGEMing.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
<br />
Team members who couldn't attend Jamboree<br />
<br />
<div class="profile"><div id="ritei" class="profilepics"></div><br />
'''Ritei Takahashi'''<br />
* School of Medicine<br />
He is also on our universities "Tori-Ninngen" team, a competition of man powered flight held every year at Lake Biwa.<br />
</div><br />
<br />
<div class="profile"><div id="kamagata" class="profilepics"></div><br />
'''Takanori Kamagata'''<br />
* Faculty of Agriculture<br />
Organizer and party spirit keeper. Helped immensely in the beginning even before iGEM team.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
----<br />
<br />
=HokkaidoU_Japan Gallery=<br />
[[Image:HokkaidoU_Japan_gallery10.jpg|165px|left|thumb|HokkaidoU_Japan Group photo]]<br />
[[Image:HokkaidoU_Japan_gallery3.jpg|165px|left|thumb|TV crew came to interview us]]<br />
[[Image:HokkaidoU_Japan_gallery1.jpg|165px|left|thumb|Graduate School of Environmental Earth Science]]<br />
[[Image:HokkaidoU_Japan_gallery2.jpg|165px|left|thumb|Hokkaido University in summer]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery8.jpg|165px|left|thumb|Hokkaido University in autumn]]<br />
[[Image:HokkaidoU_Japan_gallery9.jpg|165px|left|thumb|Hokkaido University in winter]]<br />
[[Image:HokkaidoU_Japan_gallery7.jpg|165px|left|thumb|Next year's team leader]]<br />
[[Image:HokkaidoU_Japan_gallery5.jpg|165px|left|thumb|Infection experiment]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery6.jpg|165px|left|thumb|Project leader]]<br />
[[Image:HokkaidoU_Japan_gallery11.jpg|165px|left|thumb|Discussion]]<br />
<br />
<br />
<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/d/dc/HokkaidoU_Japan_Vanagas.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/f/fb/HokkaidoU_Japan_Kamagata.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/HokkaidoU_Japan_Makino.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/HokkaidoU_Japan_Niinuma.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/0/08/HokkaidoU_Japan_Takahashi.jpg"><br />
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<img src="https://static.igem.org/mediawiki/2010/a/ac/HokkaidoU_Japan_Ritei.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/d/dc/HokkaidoU_Japan_Yamazaki.jpg"><br />
</html><br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Team
Team:HokkaidoU Japan/Team
2010-10-28T00:25:08Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<html><br />
<style><br />
<br />
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float:left;<br />
margin:5px;<br />
}<br />
<br />
</style><br />
</html><br />
=Members=<br />
[[Image:HokkaidoU_Japan_logo.png|thumb|200px|right|HokkaidoU Japan Logo]]<br />
==Instructor==<br />
<div class="profile" style="width:520px;"><div id="yamazaki" class="profilepics"></div><br />
'''Ken-ichi Yamazaki'''<br />
* Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science<br />
* [http://noah.ees.hokudai.ac.jp/emb/ymzklab/yamazaki.html Yamazaki Lab]<br />
He helped every step of the way with his advice.<br />
</div><div style="clear:both"></div><br />
<br />
==Undergrad students==<br />
<div class="profile"><div id="vanagas" class="profilepics"></div><br />
'''Laurynas Vanagas'''<br />
* Department of Biological Sciences, School of Sciences<br />
* ''Team Leader''<br />
He always comes late because of having a late clock. But it's why he works hard midnight for team members.<br />
<br />
</div><br />
<br />
<div class="profile"><div id="makino" class="profilepics"></div><br />
'''Shun-ichi Makino'''<br />
* School of Medicine<br />
* ''Project Leader''<br />
He got that crazy idea, the type 3 secreation system in E.coli.<br />
</div><br />
<br />
<div class="profile"><div id="niinuma" class="profilepics"></div><br />
'''Sho Niinuma'''<br />
* Department of Biological Sciences, School of Sciences<br />
The one who is first to come with the ideas why something was failed or why the results differ. <br />
</div><br />
<br />
<div class="profile"><div id="takahashi" class="profilepics"></div><br />
'''Kazuki Takahashi'''<br />
* Department of Biological Sciences, School of Sciences<br />
Takahashi keeps wiki in shape also responsible form team fitness and daily excersize.<br />
</div><br />
<br />
<div class="profile"><div id="matsumoto" class="profilepics"></div><br />
'''Yuichi Matsumoto'''<br />
* Department of Biological Sciences, School of Sciences<br />
In good ol' days he would go night fishing. And enjoy gourmet breakfast. But now it's night iGEMing.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
<br />
Team members who couldn't attend Jamboree<br />
<br />
<div class="profile"><div id="ritei" class="profilepics"></div><br />
'''Ritei Takahashi'''<br />
* School of Medicine<br />
He is also on our universities "Tori-Ninngen" team, a competetion of man powered flight held every year at Lake Biwa.<br />
</div><br />
<br />
<div class="profile"><div id="kamagata" class="profilepics"></div><br />
'''Takanori Kamagata'''<br />
* Faculty of Agriculture<br />
Organizer and party spirit keeper. Helped immensely in the beginning even before iGEM team.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
----<br />
<br />
=HokkaidoU_Japan Gallery=<br />
[[Image:HokkaidoU_Japan_gallery10.jpg|165px|left|thumb|HokkaidoU_Japan Group photo]]<br />
[[Image:HokkaidoU_Japan_gallery3.jpg|165px|left|thumb|TV crew came to interview us]]<br />
[[Image:HokkaidoU_Japan_gallery1.jpg|165px|left|thumb|Graduate School of Environmental Earth Science]]<br />
[[Image:HokkaidoU_Japan_gallery2.jpg|165px|left|thumb|Hokkaido University in summer]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery8.jpg|165px|left|thumb|Hokkaido University in autumn]]<br />
[[Image:HokkaidoU_Japan_gallery9.jpg|165px|left|thumb|Hokkaido University in winter]]<br />
[[Image:HokkaidoU_Japan_gallery7.jpg|165px|left|thumb|Next year's team leader]]<br />
[[Image:HokkaidoU_Japan_gallery5.jpg|165px|left|thumb|Infection experiment]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery6.jpg|165px|left|thumb|Project leader]]<br />
[[Image:HokkaidoU_Japan_gallery11.jpg|165px|left|thumb|Discussion]]<br />
<br />
<br />
<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/d/dc/HokkaidoU_Japan_Vanagas.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/f/fb/HokkaidoU_Japan_Kamagata.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/HokkaidoU_Japan_Makino.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/HokkaidoU_Japan_Niinuma.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/0/08/HokkaidoU_Japan_Takahashi.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/d/d0/HokkaidoU_Japan_Matsumoto.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/HokkaidoU_Japan_Ritei.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/d/dc/HokkaidoU_Japan_Yamazaki.jpg"><br />
</html><br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Team
Team:HokkaidoU Japan/Team
2010-10-28T00:13:32Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<html><br />
<style><br />
<br />
<br />
table .gallery td{<br />
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height:106px;<br />
float:left;<br />
margin:5px;<br />
}<br />
<br />
</style><br />
</html><br />
=Members=<br />
[[Image:HokkaidoU_Japan_logo.png|thumb|200px|right|HokkaidoU Japan Logo]]<br />
==Instructor==<br />
<div class="profile" style="width:520px;"><div id="yamazaki" class="profilepics"></div><br />
'''Ken-ichi Yamazaki'''<br />
* Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science<br />
* [http://noah.ees.hokudai.ac.jp/emb/ymzklab/yamazaki.html Yamazaki Lab]<br />
He helped every step of the way with his advice.<br />
</div><div style="clear:both"></div><br />
<br />
==Undergrad students==<br />
<div class="profile"><div id="vanagas" class="profilepics"></div><br />
'''Laurynas Vanagas'''<br />
* Department of Biological Sciences, School of Sciences<br />
* ''Team Leader''<br />
<br />
</div><br />
<br />
<div class="profile"><div id="makino" class="profilepics"></div><br />
'''Shun-ichi Makino'''<br />
* School of Medicine<br />
* ''Project Leader''<br />
He got that crazy idea, the type 3 secreation system in E.coli.<br />
</div><br />
<br />
<div class="profile"><div id="niinuma" class="profilepics"></div><br />
'''Sho Niinuma'''<br />
* Department of Biological Sciences, School of Sciences<br />
The one who is first to come with the ideas why something was failed or why the results differ. <br />
</div><br />
<br />
<div class="profile"><div id="takahashi" class="profilepics"></div><br />
'''Kazuki Takahashi'''<br />
* Department of Biological Sciences, School of Sciences<br />
Takahashi keeps wiki in shape also responsible form team fitness and daily excersize.<br />
</div><br />
<br />
<div class="profile"><div id="matsumoto" class="profilepics"></div><br />
'''Yuichi Matsumoto'''<br />
* Department of Biological Sciences, School of Sciences<br />
In good ol' days he would go night fishing every week. And enjoy gourmet breakfast. However now it's night iGEMing.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
<br />
Team members who couldn't attend Jamboree<br />
<br />
<div class="profile"><div id="ritei" class="profilepics"></div><br />
'''Ritei Takahashi'''<br />
* School of Medicine<br />
He is also on our universities "Tori-Ninngen" team, a competetion of man powered flight held every year at lake biwa.<br />
</div><br />
<br />
<div class="profile"><div id="kamagata" class="profilepics"></div><br />
'''Takanori Kamagata'''<br />
* Faculty of Agriculture<br />
Organizer and party spirit keeper. Helped immensely in the beginning even before iGEM team.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
----<br />
<br />
=HokkaidoU_Japan Gallery=<br />
[[Image:HokkaidoU_Japan_gallery10.jpg|165px|left|thumb|HokkaidoU_Japan Group photo]]<br />
[[Image:HokkaidoU_Japan_gallery3.jpg|165px|left|thumb|TV crew came to interview us]]<br />
[[Image:HokkaidoU_Japan_gallery1.jpg|165px|left|thumb|Graduate School of Environmental Earth Science]]<br />
[[Image:HokkaidoU_Japan_gallery2.jpg|165px|left|thumb|Hokkaido University in summer]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery8.jpg|165px|left|thumb|Hokkaido University in autumn]]<br />
[[Image:HokkaidoU_Japan_gallery9.jpg|165px|left|thumb|Hokkaido University in winter]]<br />
[[Image:HokkaidoU_Japan_gallery7.jpg|165px|left|thumb|Next year's team leader]]<br />
[[Image:HokkaidoU_Japan_gallery5.jpg|165px|left|thumb|Infection experiment]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery6.jpg|165px|left|thumb|Project leader]]<br />
[[Image:HokkaidoU_Japan_gallery11.jpg|165px|left|thumb|Discussion]]<br />
<br />
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<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/d/dc/HokkaidoU_Japan_Vanagas.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/f/fb/HokkaidoU_Japan_Kamagata.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/HokkaidoU_Japan_Makino.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/73/HokkaidoU_Japan_Niinuma.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/0/08/HokkaidoU_Japan_Takahashi.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/d/d0/HokkaidoU_Japan_Matsumoto.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/a/ac/HokkaidoU_Japan_Ritei.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/d/dc/HokkaidoU_Japan_Yamazaki.jpg"><br />
</html><br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Team
Team:HokkaidoU Japan/Team
2010-10-28T00:08:21Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
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=Members=<br />
[[Image:HokkaidoU_Japan_logo.png|thumb|200px|right|HokkaidoU Japan Logo]]<br />
==Instructor==<br />
<div class="profile" style="width:520px;"><div id="yamazaki" class="profilepics"></div><br />
'''Ken-ichi Yamazaki'''<br />
* Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science<br />
* [http://noah.ees.hokudai.ac.jp/emb/ymzklab/yamazaki.html Yamazaki Lab]<br />
He helped every step of the way with his advice.<br />
</div><div style="clear:both"></div><br />
<br />
==Undergrad students==<br />
<div class="profile"><div id="vanagas" class="profilepics"></div><br />
'''Laurynas Vanagas'''<br />
* Department of Biological Sciences, School of Sciences<br />
* ''Team Leader''<br />
<br />
</div><br />
<br />
<div class="profile"><div id="makino" class="profilepics"></div><br />
'''Shun-ichi Makino'''<br />
* School of Medicine<br />
* ''Project Leader''<br />
He got that crazy idea, the type 3 secreation system in E.coli.<br />
</div><br />
<br />
<div class="profile"><div id="niinuma" class="profilepics"></div><br />
'''Sho Niinuma'''<br />
* Department of Biological Sciences, School of Sciences<br />
The one who is first to come with the ideas why something was failed or why the results differ. <br />
</div><br />
<br />
<div class="profile"><div id="takahashi" class="profilepics"></div><br />
'''Kazuki Takahashi'''<br />
* Department of Biological Sciences, School of Sciences<br />
Takahashi keeps wiki in shape also responsible form team fitness and daily excersize.<br />
</div><br />
<br />
<div class="profile"><div id="matsumoto" class="profilepics"></div><br />
'''Yuichi Matsumoto'''<br />
* Department of Biological Sciences, School of Sciences<br />
In good ol' days he would go to nearly town by bicycle for night fishing every week. And enjoy gurment breakfast. However now it's night iGEMing.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
<br />
Team members who couldn't attend Jamboree<br />
<br />
<div class="profile"><div id="ritei" class="profilepics"></div><br />
'''Ritei Takahashi'''<br />
* School of Medicine<br />
He is also on our universities "Tori-Ninngen" team, a competetion of man powered flight held every year at lake biwa.<br />
</div><br />
<br />
<div class="profile"><div id="kamagata" class="profilepics"></div><br />
'''Takanori Kamagata'''<br />
* Faculty of Agriculture<br />
Organizer and party spirit keeper. Helped immensely in the beginning even before iGEM team.<br />
</div><br />
<br />
<div style="clear:both;"></div><br />
----<br />
<br />
=HokkaidoU_Japan Gallery=<br />
[[Image:HokkaidoU_Japan_gallery10.jpg|165px|left|thumb|HokkaidoU_Japan Group photo]]<br />
[[Image:HokkaidoU_Japan_gallery3.jpg|165px|left|thumb|TV crew came to interview us]]<br />
[[Image:HokkaidoU_Japan_gallery1.jpg|165px|left|thumb|Graduate School of Environmental Earth Science]]<br />
[[Image:HokkaidoU_Japan_gallery2.jpg|165px|left|thumb|Hokkaido University in summer]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery8.jpg|165px|left|thumb|Hokkaido University in autumn]]<br />
[[Image:HokkaidoU_Japan_gallery9.jpg|165px|left|thumb|Hokkaido University in winter]]<br />
[[Image:HokkaidoU_Japan_gallery7.jpg|165px|left|thumb|Next year's team leader]]<br />
[[Image:HokkaidoU_Japan_gallery5.jpg|165px|left|thumb|Infection experiment]]<br />
<div style="clear:both"></div><br />
[[Image:HokkaidoU_Japan_gallery6.jpg|165px|left|thumb|Project leader]]<br />
[[Image:HokkaidoU_Japan_gallery11.jpg|165px|left|thumb|Discussion]]<br />
<br />
<br />
<div id="preload"><br />
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<img src="https://static.igem.org/mediawiki/2010/d/dc/HokkaidoU_Japan_Vanagas.jpg"><br />
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</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan
Team:HokkaidoU Japan
2010-10-27T23:57:20Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
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<br />
----<br />
=[[Team:HokkaidoU_Japan/Projects|Projects]]=<br />
<div id="projects" class="homepics">[[Team:HokkaidoU_Japan/Projects| ]]</div><br />
==[[Team:HokkaidoU_Japan/Projects|Dr. ''E. coli'' : The smallest protein injector in the world]]==<br />
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.<br />
<br />
==[[Team:HokkaidoU_Japan/Projects/PCR|PCR Based Assembly Protocol]]==<br />
&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Team|Team]]=<br />
<div id="team" class="homepics">[[Team:HokkaidoU_Japan/Team| ]]</div><br />
&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from facaltys of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=<br />
<div id="notebook" class="homepics">[[Team:HokkaidoU_Japan/| ]]</div><br />
&nbsp;&nbsp;&nbsp;<br />
The record of our triumphs and failures. Well big part of it is failures. But we did it.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Protocols|Protocols]]=<br />
<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div><br />
&nbsp;&nbsp;&nbsp; <br />
General protocols we used.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Parts|Parts]]=<br />
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div><br />
&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.<br />
<br />
&nbsp;&nbsp;&nbsp;Also, we registerd one primer set which is useful for the PCR based protocol.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Safety|Safety]]=<br />
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div><br />
&nbsp;&nbsp;&nbsp;Addressing bio safety concerns<br />
<br />
----<br />
<br><br><br><br><br />
=Acknowledgements=<br />
==Relative to Labs==<br />
* Ken-ichi Yamazaki<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
*Hideaki Higashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Hisatoshi Shida<br />
** Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University<br />
* Takashi Oohashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Members of Yamazaki Lab<br />
*Masaaki Morikawa<br />
** Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
* Hidetoshi Okuyama<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
<br />
==Schools==<br />
* Salmonella Genetic Stock Center/University of Calgary<br />
* Graduate School of Environmental Earth Science<br />
* Department of biological science/School of Sciense<br />
* Institute for Genetic Medicine<br />
<br />
==Companies==<br />
* Amino Up Chemical<br />
* ECONIXE<br />
* Cosmo Bio<br />
* Mendel Workshop<br />
<br />
==Person==<br />
* Tadasu Emoto<br />
**Manager of ECONIXE<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="preload"><br />
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<img src="https://static.igem.org/mediawiki/2010/3/38/HokkaidoU_Japan_HomeTeam2.jpg"><br />
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</html><br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan
Team:HokkaidoU Japan
2010-10-27T23:54:56Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<html><br />
<style type="text/css"><br />
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}<br />
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<br />
----<br />
=[[Team:HokkaidoU_Japan/Projects|Projects]]=<br />
<div id="projects" class="homepics">[[Team:HokkaidoU_Japan/Projects| ]]</div><br />
==[[Team:HokkaidoU_Japan/Projects|Dr. ''E. coli'' : The smallest protein injector in the world]]==<br />
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.<br />
<br />
==[[Team:HokkaidoU_Japan/Projects/PCR|PCR Based Assembly Protocol]]==<br />
&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Team|Team]]=<br />
<div id="team" class="homepics">[[Team:HokkaidoU_Japan/Team| ]]</div><br />
&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from facaltys of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=<br />
<div id="notebook" class="homepics">[[Team:HokkaidoU_Japan/| ]]</div><br />
&nbsp;&nbsp;&nbsp;<br />
The record of our triumphs and failures. Well big part of it is failures. But we did it.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Protocols|Protocols]]=<br />
<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div><br />
&nbsp;&nbsp;&nbsp; <br />
General protocols we used.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Parts|Parts]]=<br />
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div><br />
&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.<br />
<br />
&nbsp;&nbsp;&nbsp;Also, we registerd one primer set which is useful for the PCR based protocol.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Safety|Safety]]=<br />
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div><br />
&nbsp;&nbsp;&nbsp;Addressing bio safety concerns<br />
<br />
----<br />
<br><br><br><br><br />
=Acknowledgements=<br />
==Relative to Labs==<br />
* Ken-ichi Yamazaki<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
*Hideaki Higashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Hisatoshi Shida<br />
** Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University<br />
* Takashi Oohashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Members of Yamazaki Lab<br />
*Masaaki Morikawa<br />
** Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
* Hidetoshi Okuyama<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
<br />
==Schools==<br />
* University of Calgary<br />
* Graduate School of Environmental Earth Science<br />
* Department of biological science/School of Sciense<br />
* Institute for Genetic Medicine<br />
<br />
==Companies==<br />
* Amino Up Chemical<br />
* ECONIXE<br />
* Cosmo Bio<br />
* Mendel Workshop<br />
<br />
==Others==<br />
* Salmonella Genetic Stock Center/University of Calgary<br />
* Tadasu Emoto<br />
**Manager of ECONIXE<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/3/38/HokkaidoU_Japan_HomeTeam2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/2/2a/HokkaidoU_Japan_HomeSafety2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a2/HokkaidoU_Japan_HomeProject2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7c/HokkaidoU_Japan_HomeNotebook2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a4/HokkaidoU_Japan_HomeParts2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ec/HokkaidoU_Japan_HomeProtocols2.jpg"><br />
</html><br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan
Team:HokkaidoU Japan
2010-10-27T23:47:49Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
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<br />
----<br />
=[[Team:HokkaidoU_Japan/Projects|Projects]]=<br />
<div id="projects" class="homepics">[[Team:HokkaidoU_Japan/Projects| ]]</div><br />
==[[Team:HokkaidoU_Japan/Projects|Dr. ''E. coli'' : The smallest protein injector in the world]]==<br />
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.<br />
<br />
==[[Team:HokkaidoU_Japan/Projects/PCR|PCR Based Assembly Protocol]]==<br />
&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Team|Team]]=<br />
<div id="team" class="homepics">[[Team:HokkaidoU_Japan/Team| ]]</div><br />
&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from facaltys of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=<br />
<div id="notebook" class="homepics">[[Team:HokkaidoU_Japan/| ]]</div><br />
&nbsp;&nbsp;&nbsp;<br />
The record of our triumphs and failures. Well big part of it is failures. But we did it.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Protocols|Protocols]]=<br />
<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div><br />
&nbsp;&nbsp;&nbsp; <br />
General protocols we used.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Parts|Parts]]=<br />
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div><br />
&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.<br />
<br />
&nbsp;&nbsp;&nbsp;Also, we registerd one primer set which is useful for the PCR based protocol.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Safety|Safety]]=<br />
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div><br />
&nbsp;&nbsp;&nbsp;Addressing bio safety concerns<br />
<br />
----<br />
<br><br><br><br><br />
=Acknowledgements=<br />
==Relative to Labs==<br />
* Ken-ichi Yamazaki<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
*Hideaki Higashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Hisatoshi Shida<br />
** Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University<br />
* Takashi Oohashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Members of Yamazaki Lab<br />
*Masaaki Morikawa<br />
** Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
* Hidetoshi Okuyama<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
<br />
==Schools==<br />
* Graduate School of Environmental Earth Science<br />
* Department of biological science/School of Sciense<br />
* Institute for Genetic Medicine<br />
<br />
==Companies==<br />
* Amino Up Chemical<br />
* ECONIXE<br />
* Cosmo Bio<br />
* Mendel Workshop<br />
<br />
==Others==<br />
* Salmonella Genetic Stock Center<br />
* Tadasu Emoto<br />
**Manager of ECONIXE<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/3/38/HokkaidoU_Japan_HomeTeam2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/2/2a/HokkaidoU_Japan_HomeSafety2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a2/HokkaidoU_Japan_HomeProject2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7c/HokkaidoU_Japan_HomeNotebook2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a4/HokkaidoU_Japan_HomeParts2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/e/ec/HokkaidoU_Japan_HomeProtocols2.jpg"><br />
</html><br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan
Team:HokkaidoU Japan
2010-10-27T23:46:34Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<html><br />
<style type="text/css"><br />
hr {<br />
clear:both;<br />
}<br />
<br />
#main p{<br />
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}<br />
</style><br />
</html><br />
<br />
----<br />
=[[Team:HokkaidoU_Japan/Projects|Projects]]=<br />
<div id="projects" class="homepics">[[Team:HokkaidoU_Japan/Projects| ]]</div><br />
==[[Team:HokkaidoU_Japan/Projects|Dr. ''E. coli'' : The smallest protein injector in the world]]==<br />
&nbsp;&nbsp;&nbsp;Our main project is on Type III Secretion Apparatus which is one of the most amazing biological devices. This apparatus which looks like a syringe can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. However, this apparatus is an organelle of pathogenic gram-negative bacterium such as ''Salmonella'' and ''Yersinia''. So we are aiming at making this device safely available using ''E. coli''.<br />
<br />
==[[Team:HokkaidoU_Japan/Projects/PCR|PCR Based Assembly Protocol]]==<br />
&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't be amplified. In moments like these PCR is handy. We think we might slightly improved it. You no longer have to guess if restriction was successful.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Team|Team]]=<br />
<div id="team" class="homepics">[[Team:HokkaidoU_Japan/Team| ]]</div><br />
&nbsp;&nbsp;&nbsp;The Hokkaido University's igem 2010 team, HokkaidoU_Japan is Japans 8th Our has one instructer and 7 undergrad students.They come from facaltys of Sciences, Medicine and Agriculture. This is the first year we are participating in iGEM.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Notebook|Notebook]]=<br />
<div id="notebook" class="homepics">[[Team:HokkaidoU_Japan/| ]]</div><br />
&nbsp;&nbsp;&nbsp;<br />
The record of our triumphs and failures. Well big part of it is failures. But we did it.<br />
<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Protocols|Protocols]]=<br />
<div id="protocols" class="homepics">[[Team:HokkaidoU_Japan/Protocols| ]]</div><br />
&nbsp;&nbsp;&nbsp; <br />
General protocols we used.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Parts|Parts]]=<br />
<div id="parts" class="homepics">[[Team:HokkaidoU_Japan/Parts| ]]</div><br />
&nbsp;&nbsp;&nbsp;We registered two parts associated with main project. One is a signal sequence that can be used to secrete tagged proteins through Type III Secretion Apparatus. The other is whole reporter construct to checks whether the Type III secretion system works correctly.<br />
<br />
&nbsp;&nbsp;&nbsp;Also, we registerd one primer set which is useful for the PCR based protocol.<br />
----<br />
<br />
=[[Team:HokkaidoU_Japan/Safety|Safety]]=<br />
<div id="safety" class="homepics">[[Team:HokkaidoU_Japan/Safety| ]]</div><br />
&nbsp;&nbsp;&nbsp;Addressing bio safety concerns<br />
<br />
----<br />
<br><br><br><br><br />
=Acknowledgements=<br />
==Relative to Labs==<br />
* Ken-ichi Yamazaki<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaido University<br />
*Hideaki Higashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Hisatoshi Shida<br />
** Professor/Molecular Biology/Institute for Genetic Medicine/ Hokkaido University<br />
* Takashi Oohashi<br />
** Associate Professor /Division of Molecular Oncology/ Institute for Genetic Medicine/ Hokkaido University<br />
* Members of Yamazaki Lab<br />
*Masaaki Morikawa<br />
** Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
* Hidetoshi Okuyama<br />
** Associate Professor/Environmental Molecular Biology/Graduate School of Environmental Earth Science/Hokkaidou University<br />
<br />
==Schools==<br />
* Graduate School of Environmental Earth Science<br />
* Department of biological science/School of Sciense<br />
* Institute for Genetic Medicine<br />
<br />
==Companies==<br />
* Amino Up Chemical<br />
* ECONIXE<br />
* Cosmo Bio<br />
* Mendel Workshop<br />
<br />
==Others==<br />
* Salmonera Genetic Stock Center<br />
* Tadasu Emoto<br />
**Manager of ECONIXE<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<div id="preload"><br />
<html><br />
<img src="https://static.igem.org/mediawiki/2010/3/38/HokkaidoU_Japan_HomeTeam2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/2/2a/HokkaidoU_Japan_HomeSafety2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/a/a2/HokkaidoU_Japan_HomeProject2.jpg"><br />
<img src="https://static.igem.org/mediawiki/2010/7/7c/HokkaidoU_Japan_HomeNotebook2.jpg"><br />
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<img src="https://static.igem.org/mediawiki/2010/e/ec/HokkaidoU_Japan_HomeProtocols2.jpg"><br />
</html><br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September23
Team:HokkaidoU Japan/Notebook/September23
2010-10-27T18:02:42Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September22|September 22]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September24|September 24]]</div></div><br />
<br />
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification<br />
*Colony PCR of AraC+RBS+pSB1A3<br />
*Electroporetion of BAC plasmid into DH5α MG1655<br />
<br />
= Bac Vecter Purification =<br />
<br />
Used Qiagen miniprep kit, qiaprep for miniprep<br />
<br />
#Transfered 1.3 mL of BAC plasmid solution incubated overnight into 1.5 mL tube<br />
#Centrifuged at 4C, 15000 rpm for 1 min<br />
#Discarded the supernatant and added remaining solution.<br />
#Centrifuged at 4C, 15000 rpm for 1 min<br />
#Discarded the supernatant<br />
#Suspended on 250 uL of Buffer P1<br />
#Added 250 ul Buffer P2 inverted few times to mix, solution turned green<br />
#Added 350 ul Buffer N3 mixed by inversion, precipitation apeared<br />
#Centrifuged at 4C, 13000 rpm for 10 min<br />
#Transfered the supernatant to filtration column <br />
#Centrifuged at 4C, 13000 rpm for 1 min<br />
#Discarded the flow-through<br />
#added 500 uL of Buffer PB to filtration column <br />
#Centrifuged at 4C, 13000 rpm for 1 min<br />
#Discarded the flow-through centrifuged for 1min to remove remaining buffer<br />
#Transfered filtration column to a new 1.5 ml tube<br />
#Resuspended on 50 ul of TE and incubated at RT for 1min<br />
#Centrifuged at 4C, 13000 rpm for 1 min<br />
#Stored at -20C<br />
<br />
<br />
= Colony PCR of AraC+RBS+pSB1A3 =<br />
*Selected 16 colonies at random, and PCRed them. <br />
*PCR mix used is shown in the table bellow<br />
<br />
{|style="text-align:center; margin-left:100px;" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Taq Master Mix<br />
|80 uL<br />
|-<br />
|Ex-F<br />
|1.6 uL<br />
|-<br />
|Ps-R<br />
|1.6 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''83.2 uL'''<br />
|}<br />
<br />
[[Image:9.23 coloP.JPG|200px|right|thumb|Result of colony PCR]]<br />
<br />
*electrophoresed the samples<br />
<br />
1 lane -> Marker λ/HindIII EcoRI 5 uL<br />
<br />
2~8 lane -> colony No.1~7<br />
<br />
9 lane -> Marker λ/HindIII EcoRI 5 uL<br />
<br />
10~16 lane -> colony No.8~14<br />
<br />
<br />
Colony No.15,16 didn't electrophorese.<br />
<br />
<br />
*Colony No.1 will have the plasmid,so we put the sample into 2 mL LB added 2 uL Ampicillin(100 ug/uL).Incubated it in a shaker at 37C, 180 rpm.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September21
Team:HokkaidoU Japan/Notebook/September21
2010-10-27T18:02:11Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September20|September 20]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September22|September 22]]</div></div><br />
<br />
<br />
== Annealing of RBS made from oligos ==<br />
<br />
[[Image:HokkaidoU_Japan_RBS_B0034.png|200px|right|frame| Construction of RBS (BBa_B0034) from oligos. Already precut!]]<br />
Annealing of RBS (BBa_B0034) made from oligos<br />
<br />
For this I ordered oligo primers for sigma<br />
<br><br><br><br><br><br />
== アラビノース依存型GFP発現装置の作成 ==<br />
パーツ情報<br />
*GFP-doubleterminator<br />
BBa_I13401 1-12K 875bp pSB1A2<br />
*アラビノースプロモーター<br />
BBa_I0050 3-20B 1210bp pSB2K3<br />
*pSB1C3 2072bp<br />
*pSB1A3 2157bp<br />
<br />
<br><br />
<br />
1)アラビノースプロモーター、GFP、pSB1A3を以下の組成で調整した。<br />
{|style="text-align:center; margin-left:100px;" class="protocol" <br />
!Reagent<br />
!Amount<br />
|-<br />
|Dw<br />
|33 uL<br />
|-<br />
|10×PCR buffur<br />
|5<br />
|-<br />
|2mM 4dNTP<br />
|5<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|Ex-F<br />
|1<br />
|-<br />
|Ps-R<br />
|1<br />
|-<br />
|KOD neo plus<br />
|1<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}<br />
<br />
2)<br />
<br />
94C 2min<br />
<br />
98C 10sec<br />
<br />
68C 2min<br />
<br />
35cycle<br />
<br />
4C hold<br />
<br />
という設定でPCRした。<br />
<br />
<br />
3)PCR後、10 ulのローディングバッファーを各サンプルに入れて1レーンにつき12 ul入れ泳動した。<br />
<br />
<br />
4)サンプルをゲルから抽出し、promegaのキットを用いて精製した。<br />
<br />
<br />
5)精製したサンプルを1 ul取り、1 ulのローディングバッファーを加えた後、6 ulのλ/Hind Ⅲ EcoRⅠとともに泳動した。<br />
<br />
<div style="margin-left:100px;"><br />
[[Image:HokkaidoU_Japan_20100921a.JPG|200px|thumb|left|泳動結果]]<br />
<br />
<br />
左から<br />
<br />
1.λ/Hind Ⅲ EcoRⅠ<br />
<br />
2.アラビノースプロモーター<br />
<br />
3.GFP+double terminator<br />
<br />
4.pSB1A3<br />
<br />
5.RFP reporter<br />
<br />
6.RFP?<br />
<br />
7.Heat shock promoter</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook
Team:HokkaidoU Japan/Notebook
2010-10-27T18:01:20Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
__NOTOC__<br />
<html><br />
<style><br />
#main h3{<br />
font-size: 116%; <br />
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}<br />
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background-color:#F1F0E7;<br />
text-align:center;<br />
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width:180px;<br />
}<br />
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.calendar a {<br />
display: block;<br />
font-weight: bold;<br />
text-decoration: none;<br />
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</style><br />
</html><br />
==Calendar==<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|July<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
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|2<br />
|style="color:blue;"|3<br />
|-<br />
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|6<br />
|7<br />
|8<br />
|9<br />
|style="color:blue;"|10<br />
|-<br />
|style="color:red;"|11<br />
|[[#Monday, July 12|12]]<br />
|13<br />
|14<br />
|15<br />
|16<br />
|style="color:blue;"|17<br />
|-<br />
|style="color:red;"|18<br />
|19<br />
|20<br />
|[[#Wednesday, July 21|21]]<br />
|22<br />
|23<br />
|style="color:blue;"|24<br />
|-<br />
|style="color:red;"|25<br />
|[[#Monday, July 26|26]]<br />
|[[#Tuesday, July 27|27]]<br />
|28<br />
|29<br />
|30<br />
|style="color:blue;"|31<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|August<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|1<br />
|[[#Monday, August 2|2]]<br />
|3<br />
|4<br />
|5<br />
|6<br />
|style="color:blue;"|7<br />
|-<br />
|style="color:red;"|8<br />
|[[#Monday, August 9|9]]<br />
|[[#Tuesday, August 10|10]]<br />
|[[#Wednesday, August 11|11]]<br />
|[[#Thursday, August 12|12]]<br />
|[[#Friday, August 13|13]]<br />
|style="color:blue;"|14<br />
|-<br />
|style="color:red;"|15<br />
|[[#Monday, August 16|16]]<br />
|[[#Tuesday, August 17|17]]<br />
|[[#Wednesday, August 18|18]]<br />
|[[#Thursday, August 19|19]]<br />
|[[#Friday, August 20|20]]<br />
|style="color:blue;"|21<br />
|-<br />
|style="color:red;"|22<br />
|[[#Monday, August 23|23]]<br />
|[[#Tuesday, August 24|24]]<br />
|[[#Wednesday, August 25|25]]<br />
|[[#Thursday, August 26|26]]<br />
|[[#Friday, August 27|27]]<br />
|style="color:blue;"|28<br />
|-<br />
|style="color:red;"|29<br />
|[[#Monday, August 30|30]]<br />
|[[#Tuesday, August 31|31]]<br />
|<br />
|<br />
| <br />
|style="color:blue;"|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|September<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|[[#Wednesday, September 1|1]]<br />
|[[#Thursday, September 2|2]]<br />
|[[#Friday, September 3|3]]<br />
|style="color:blue;"|[[#Saturday, September 4|4]]<br />
|-<br />
|style="color:red;"|5<br />
|[[#Monday, September 6|6]]<br />
|[[#Tuesday, September 7|7]]<br />
|[[#Wednesday, September 8|8]]<br />
|[[#Thursday, September 9|9]]<br />
|[[#Friday, September 10|10]]<br />
|style="color:blue;"|11<br />
|-<br />
|style="color:red;"|12<br />
|[[#Monday, September 13|13]]<br />
|[[#Tuesday, September 14|14]]<br />
|[[#Wednesday, September 15|15]]<br />
|[[#Thursday, September 16|16]]<br />
|[[#Friday, September 17|17]]<br />
|style="color:blue;"|18<br />
|-<br />
|style="color:red;"|19<br />
|20<br />
|[[#Tuesday, September 21|21]]<br />
|[[#Wednesday, September 22|22]]<br />
|[[#Thursday, September 23|23]]<br />
|[[#Friday, September 24|24]]<br />
|style="color:blue;"|25<br />
|-<br />
|style="color:red;"|26<br />
|[[#Monday, September 27|27]]<br />
|[[#Tuesday, September 28|28]]<br />
|[[#Wednesday, September 29|29]]<br />
|[[#Thursday, September 30|30]]<br />
|<br />
|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|October<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|1<br />
|style="color:blue;"|[[#Saturday, October 2|2]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 3|3]]<br />
|[[#Monday, October 4|4]]<br />
|[[#Tuesday, October 5|5]]<br />
|[[#Wednesday, October 6|6]]<br />
|[[#Thursday, October 7|7]]<br />
|[[#Friday, October 8|8]]<br />
|style="color:blue;"|[[#Saturday, October 9|9]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 10|10]]<br />
|[[#Monday, October 11|11]]<br />
|[[#Tuesday, October 12|12]]<br />
|[[#Wednesday, October 13|13]]<br />
|[[#Thursday, October 14|14]]<br />
|[[#Friday, October 15|15]]<br />
|style="color:blue;"|[[#Saturday, October 16|16]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 17|17]]<br />
|[[#Monday, October 18|18]]<br />
|[[#Tuesday, October 19|19]]<br />
|[[#Wednesday, October 20|20]]<br />
|[[#Thursday, October 21|21]]<br />
|[[#Friday, October 22|22]]<br />
|style="color:blue;"|[[#Saturday, October 23|23]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 24|24]]<br />
|[[#Monday, October 25|25]]<br />
|[[#Tuesday, October 26|26]]<br />
|[[#Wednesday, October 27|27]]<br />
|[[#Thursday, October 28|28]]<br />
|[[#Friday, October 29|29]]<br />
|style="color:blue;"|[[#Saturday, October 30|30]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 31|31]]<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:blue;"|<br />
|}<br />
<div style="clear:both"></div><br />
<br />
==Abstract==<br />
===Monday, July 12===<br />
* Our first experiment, transformation of BioBrick devices.<br />
----<br />
<br />
===Wednesday, July 21===<br />
* Preparation of competent cells<br />
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12<br />
<br />
===Monday, July 26===<br />
* Cultivation of transformed ''E. coli'' for the next day's miniprep<br />
<br />
===Tuesday, July 27===<br />
* Miniprep (Alkaline SDS method)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August2|Monday, August 2]]===<br />
* Restriction enzyme digestion of plasmids which had been purified by miniprep<br />
* Electrophoresis assay<br />
<br />
===Tuesday, August 3===<br />
===Wednesday, August 4===<br />
===Thursday, August 5===<br />
===Friday, August 6===<br />
----<br />
===Monday, August 9===<br />
* Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive ''E. coli and heat-sensitive ''E. coli<br />
* These are preliminary experiments before starting the main project<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August10|Tuesday, August 10]]===<br />
* PCR amplification of the other parts (BBa_F1610 "2-24G", BBa_B0034 "1-2M", BBa_B0015 "1-23L" and BBa_K098995 "3-1E") and electrophoresis to confirm<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August11|Wednesday, August 11]]===<br />
* Preparation for the next day's miniprep<br />
* Estimation of enzyme activity<br />
* Preparation of the glycerol stocks<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August12|Thursday, August 12]]===<br />
* Miniprep (1-18F, 2-21H and 2-11P)<br />
* Electrophoresis of the DNA which had been amplified via PCR and miniprep<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August13|Friday, August 13]]===<br />
* PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August16|Monday, August 16]]===<br />
* Measurement of restriction enzyme activity<br />
* Restriction enzyme digestion of the parts(3-1E, 1-2M, 1-18F and 1-23L) and the vector(pSB1C3)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August17|Tuesday, August 17]]===<br />
* Purification of the DNA solutions via gel extraction<br />
* Preparation of agar medium containing 35 ug/mL chloramphenicol<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August18|Wednesday, August 18]]===<br />
* Digestion and gel extraction of 1-2M (retry)<br />
* 3 piece ligation of 1-18F, 1-23L and pSB1C3<br />
* Transformation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August19|Thursday, August 19]]===<br />
* 3 piece ligation of 3-1E (heat sensor), 1-2M (ribosome binding site) and pSB1C3 (vector)<br />
* Ligation that uses only vector pSB1C3 to estimate its efficiency<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August20|Friday, August 20]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August23|Monday, August 23]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)<br />
* PCR amplification of BioBrick parts using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August24|Tuesday, August 24]]===<br />
* Electrophoresis of PCR products that had been amplified using new primers<br />
* Examination of two ligation kits<br />
* Electrophoresis assay of miscellaneous DNA solutions<br />
* Transformation of 1-3A (RFP reporter with chloramphenicol resistance) to evaluate the medium<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August25|Wednesday, August 25]]===<br />
* Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium<br />
* Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation<br />
* Digestion of PCR products that had been amplified by using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August26|Thursday, August 26]]===<br />
* Electriphoresis to check whether ligation had been successful<br />
* Digestion of PCR products that had been amplified using new primers (with reconstruction of reaction system)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August27|Friday, August 27]]===<br />
* Ligation of 1-1A (RFP reporter device) into pSB1C3 and its transformation<br />
* Retry of 3 piece ligation which was done on August 19th and 20th<br />
* Ligation of vectors to each other<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August30|Monday, August 30]]===<br />
* Measurement of restriction enzyme activity using highly purified pUC119<br />
* Digestion of PCR products that had been amplified using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August31|Tuesday, August 31]]===<br />
* Gel extraction, ligation and transformation of pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September1|Wednesday, September 1]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September2|Thursday, September 2]]===<br />
* Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September3|Friday, September 3]]===<br />
* Colony PCR of ''E. coli'' transformed on previous day<br />
* PCR amplification of 1-3A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September4|Saturday, September 4]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
* Estimation of the amount of 1-3A PCR product<br />
<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September6|Monday, September 6]]===<br />
*Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September7|Tuesday, September 7]]===<br />
*Concentration check of DNA used for ligation yesterday<br />
*Ethanol precipitation<br />
*Colony PCR of competent cells<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September8|Wednesday, September 8]]===<br />
*Colony PCR<br />
*Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3<br />
*PCR of GFP<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September9|Thursday, September 9]]===<br />
* 3 piece ligation of HSP, GFP and pSB1C3<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]===<br />
*3 piece ligation, continued<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September13|Monday, September 13]]===<br />
* AraC promoter purification<br />
* And follow up checks for quality<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September14|Tuesday, September 14]]===<br />
* Digestion and ligation of pSB1C3, araC Promoter and GFP<br />
* Ethanol precipitation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September15|Wednesday, September 15]]===<br />
*Observed results of yesterdays transformation<br />
**Transformation using heat shock went well<br />
**Electroporation transformation failed produce colonies<br />
*Did Colony PCR of yesterdays transformed colonies<br />
*Introduced colonies to L(+)Arabinose medium to check if it would show desired function<br />
**Check for results tomorrow<br />
<br />
===Thursday, September 16===<br />
*Observed results of overnight incubation<br />
**Fluorescence was visible when viewed by fluorescence microscope<br />
*Did experiment to see if fluorescence is affected by arabinose concentration<br />
*Scanned GFP intensity of broth containing colonies we isolated yesterday<br />
*Had a free time so amplified some parts for easy training constructs<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September17|Friday, September 17]]===<br />
*Construction of GFP marker for a part which will be secreted using T3SS<br />
*Ordered primers for construction for same part<br />
----<br />
<br />
===Monday, September 20===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]===<br />
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September22|Thursday, September 22]]===<br />
*Digestion of pSB1A3, Arabinose promoter and GFP + double terminator<br />
*Ligation & Transformation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]===<br />
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification<br />
*Colony PCR of AraC+RBS+pSB1A3<br />
*Electroporetion of BAC plasmid into DH5α MG1655<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September24|Friday, September 24]]===<br />
*Miniprep of Arac+RBS+pSB1A3<br />
*Follow quality check<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===<br />
* Culture of the BAC clones<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]===<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September29|Wednesday, September 29]]===<br />
*Electrophoresis of T3SS signal and GFP + double terminator<br />
*Colony PCR and cultivation of arabinose promoter + RBS + GFP + double terminator<br />
*making competent cell of E.coli with SPI2<br />
*PCR of T3SS signal Again<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]===<br />
*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3<br />
*Transformation<br />
<br />
===Friday, October 1===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]===<br />
* Colony PCR<br />
* Preparation for Sequencing<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]===<br />
* Miniprep<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October6|Wednesday, October 6]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October9|Saturday, October 9]]===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October12|Tuesday, October 12]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October13|Wednesday, October 13]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October16|Saturday, October 16]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October17|Sunday, October 17]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October19|Tuesday, October 19]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October20|Wednesday, October 20]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October23|Saturday, October 23]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October24|Sunday, October 24]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October26|Tuesday, October 26]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October27|Wednesday, October 27]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October30|Saturday, October 30]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October31|Sunday, October 31]]===</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September22
Team:HokkaidoU Japan/Notebook/September22
2010-10-27T17:57:11Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September21|September 21]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September23|September 23]]</div></div><br />
<br />
= Digestion of pSB1A3, Arabinose Promoter & GFP + double terminator =<br />
<br />
*Digestion mix -> XbaI in GFP + double terminator solution required putting on incubator for a long time, so did it. <br />
<br />
{|style="text-align:center; float:left;" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|10x H buffer<br />
|2 uL<br />
|-<br />
|DW<br />
|10<br />
|-<br />
|pSB1A3<br />
|15<br />
|-<br />
|0.1% BSA<br />
|2<br />
|-<br />
|EcoR I<br />
|0.5<br />
|-<br />
|Pst I<br />
|0.5<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''30 uL'''<br />
|}<br />
{|style="text-align:center; float:left;" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|10x H buffer<br />
|2 uL<br />
|-<br />
|DW<br />
|8<br />
|-<br />
|Arabinose Promoter<br />
|6.3<br />
|-<br />
|0.1% BSA<br />
|2<br />
|-<br />
|EcoR I<br />
|1<br />
|-<br />
|Spe I<br />
|0.7<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''20 uL'''<br />
|}<br />
{|style="text-align:center; float:left;" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|10x M buffer<br />
|5 uL<br />
|-<br />
|DW<br />
|32.5<br />
|-<br />
|GFP + double terminator<br />
|3<br />
|-<br />
|BSA<br />
|5<br />
|-<br />
|Xba I<br />
|4<br />
|-<br />
|Pst I<br />
|0.5<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}<br />
<div style="clear:both"></div><br />
<br />
*Precipitated two samples by EtOH after extracted them from a gel.<br />
->Ligation & Transformation</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook
Team:HokkaidoU Japan/Notebook
2010-10-27T17:35:15Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
__NOTOC__<br />
<html><br />
<style><br />
#main h3{<br />
font-size: 116%; <br />
font-weight:normal;<br />
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#main ul {<br />
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text-align:center;<br />
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.calendar a {<br />
display: block;<br />
font-weight: bold;<br />
text-decoration: none;<br />
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<br />
</style><br />
</html><br />
==Calendar==<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|July<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|<br />
|1<br />
|2<br />
|style="color:blue;"|3<br />
|-<br />
|style="color:red;"|4<br />
|5<br />
|6<br />
|7<br />
|8<br />
|9<br />
|style="color:blue;"|10<br />
|-<br />
|style="color:red;"|11<br />
|[[#Monday, July 12|12]]<br />
|13<br />
|14<br />
|15<br />
|16<br />
|style="color:blue;"|17<br />
|-<br />
|style="color:red;"|18<br />
|19<br />
|20<br />
|[[#Wednesday, July 21|21]]<br />
|22<br />
|23<br />
|style="color:blue;"|24<br />
|-<br />
|style="color:red;"|25<br />
|[[#Monday, July 26|26]]<br />
|[[#Tuesday, July 27|27]]<br />
|28<br />
|29<br />
|30<br />
|style="color:blue;"|31<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|August<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|1<br />
|[[#Monday, August 2|2]]<br />
|3<br />
|4<br />
|5<br />
|6<br />
|style="color:blue;"|7<br />
|-<br />
|style="color:red;"|8<br />
|[[#Monday, August 9|9]]<br />
|[[#Tuesday, August 10|10]]<br />
|[[#Wednesday, August 11|11]]<br />
|[[#Thursday, August 12|12]]<br />
|[[#Friday, August 13|13]]<br />
|style="color:blue;"|14<br />
|-<br />
|style="color:red;"|15<br />
|[[#Monday, August 16|16]]<br />
|[[#Tuesday, August 17|17]]<br />
|[[#Wednesday, August 18|18]]<br />
|[[#Thursday, August 19|19]]<br />
|[[#Friday, August 20|20]]<br />
|style="color:blue;"|21<br />
|-<br />
|style="color:red;"|22<br />
|[[#Monday, August 23|23]]<br />
|[[#Tuesday, August 24|24]]<br />
|[[#Wednesday, August 25|25]]<br />
|[[#Thursday, August 26|26]]<br />
|[[#Friday, August 27|27]]<br />
|style="color:blue;"|28<br />
|-<br />
|style="color:red;"|29<br />
|[[#Monday, August 30|30]]<br />
|[[#Tuesday, August 31|31]]<br />
|<br />
|<br />
| <br />
|style="color:blue;"|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|September<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|[[#Wednesday, September 1|1]]<br />
|[[#Thursday, September 2|2]]<br />
|[[#Friday, September 3|3]]<br />
|style="color:blue;"|[[#Saturday, September 4|4]]<br />
|-<br />
|style="color:red;"|5<br />
|[[#Monday, September 6|6]]<br />
|[[#Tuesday, September 7|7]]<br />
|[[#Wednesday, September 8|8]]<br />
|[[#Thursday, September 9|9]]<br />
|[[#Friday, September 10|10]]<br />
|style="color:blue;"|11<br />
|-<br />
|style="color:red;"|12<br />
|[[#Monday, September 13|13]]<br />
|[[#Tuesday, September 14|14]]<br />
|[[#Wednesday, September 15|15]]<br />
|[[#Thursday, September 16|16]]<br />
|[[#Friday, September 17|17]]<br />
|style="color:blue;"|18<br />
|-<br />
|style="color:red;"|19<br />
|20<br />
|[[#Tuesday, September 21|21]]<br />
|[[#Wednesday, September 22|22]]<br />
|[[#Thursday, September 23|23]]<br />
|[[#Friday, September 24|24]]<br />
|style="color:blue;"|25<br />
|-<br />
|style="color:red;"|26<br />
|[[#Monday, September 27|27]]<br />
|[[#Tuesday, September 28|28]]<br />
|[[#Wednesday, September 29|29]]<br />
|[[#Thursday, September 30|30]]<br />
|<br />
|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|October<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|1<br />
|style="color:blue;"|[[#Saturday, October 2|2]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 3|3]]<br />
|[[#Monday, October 4|4]]<br />
|[[#Tuesday, October 5|5]]<br />
|[[#Wednesday, October 6|6]]<br />
|[[#Thursday, October 7|7]]<br />
|[[#Friday, October 8|8]]<br />
|style="color:blue;"|[[#Saturday, October 9|9]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 10|10]]<br />
|[[#Monday, October 11|11]]<br />
|[[#Tuesday, October 12|12]]<br />
|[[#Wednesday, October 13|13]]<br />
|[[#Thursday, October 14|14]]<br />
|[[#Friday, October 15|15]]<br />
|style="color:blue;"|[[#Saturday, October 16|16]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 17|17]]<br />
|[[#Monday, October 18|18]]<br />
|[[#Tuesday, October 19|19]]<br />
|[[#Wednesday, October 20|20]]<br />
|[[#Thursday, October 21|21]]<br />
|[[#Friday, October 22|22]]<br />
|style="color:blue;"|[[#Saturday, October 23|23]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 24|24]]<br />
|[[#Monday, October 25|25]]<br />
|[[#Tuesday, October 26|26]]<br />
|[[#Wednesday, October 27|27]]<br />
|[[#Thursday, October 28|28]]<br />
|[[#Friday, October 29|29]]<br />
|style="color:blue;"|[[#Saturday, October 30|30]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 31|31]]<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:blue;"|<br />
|}<br />
<div style="clear:both"></div><br />
<br />
==Abstract==<br />
===Monday, July 12===<br />
* Our first experiment, transformation of BioBrick devices.<br />
----<br />
<br />
===Wednesday, July 21===<br />
* Preparation of competent cells<br />
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12<br />
<br />
===Monday, July 26===<br />
* Cultivation of transformed ''E. coli'' for the next day's miniprep<br />
<br />
===Tuesday, July 27===<br />
* Miniprep (Alkaline SDS method)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August2|Monday, August 2]]===<br />
* Restriction enzyme digestion of plasmids which had been purified by miniprep<br />
* Electrophoresis assay<br />
<br />
===Tuesday, August 3===<br />
===Wednesday, August 4===<br />
===Thursday, August 5===<br />
===Friday, August 6===<br />
----<br />
===Monday, August 9===<br />
* Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive ''E. coli and heat-sensitive ''E. coli<br />
* These are preliminary experiments before starting the main project<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August10|Tuesday, August 10]]===<br />
* PCR amplification of the other parts (BBa_F1610 "2-24G", BBa_B0034 "1-2M", BBa_B0015 "1-23L" and BBa_K098995 "3-1E") and electrophoresis to confirm<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August11|Wednesday, August 11]]===<br />
* Preparation for the next day's miniprep<br />
* Estimation of enzyme activity<br />
* Preparation of the glycerol stocks<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August12|Thursday, August 12]]===<br />
* Miniprep (1-18F, 2-21H and 2-11P)<br />
* Electrophoresis of the DNA which had been amplified via PCR and miniprep<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August13|Friday, August 13]]===<br />
* PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August16|Monday, August 16]]===<br />
* Measurement of restriction enzyme activity<br />
* Restriction enzyme digestion of the parts(3-1E, 1-2M, 1-18F and 1-23L) and the vector(pSB1C3)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August17|Tuesday, August 17]]===<br />
* Purification of the DNA solutions via gel extraction<br />
* Preparation of agar medium containing 35 ug/mL chloramphenicol<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August18|Wednesday, August 18]]===<br />
* Digestion and gel extraction of 1-2M (retry)<br />
* 3 piece ligation of 1-18F, 1-23L and pSB1C3<br />
* Transformation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August19|Thursday, August 19]]===<br />
* 3 piece ligation of 3-1E (heat sensor), 1-2M (ribosome binding site) and pSB1C3 (vector)<br />
* Ligation that uses only vector pSB1C3 to estimate its efficiency<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August20|Friday, August 20]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August23|Monday, August 23]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)<br />
* PCR amplification of BioBrick parts using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August24|Tuesday, August 24]]===<br />
* Electrophoresis of PCR products that had been amplified using new primers<br />
* Examination of two ligation kits<br />
* Electrophoresis assay of miscellaneous DNA solutions<br />
* Transformation of 1-3A (RFP reporter with chloramphenicol resistance) to evaluate the medium<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August25|Wednesday, August 25]]===<br />
* Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium<br />
* Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation<br />
* Digestion of PCR products that had been amplified by using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August26|Thursday, August 26]]===<br />
* Electriphoresis to check whether ligation had been successful<br />
* Digestion of PCR products that had been amplified using new primers (with reconstruction of reaction system)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August27|Friday, August 27]]===<br />
* Ligation of 1-1A (RFP reporter device) into pSB1C3 and its transformation<br />
* Retry of 3 piece ligation which was done on August 19th and 20th<br />
* Ligation of vectors to each other<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August30|Monday, August 30]]===<br />
* Measurement of restriction enzyme activity using highly purified pUC119<br />
* Digestion of PCR products that had been amplified using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August31|Tuesday, August 31]]===<br />
* Gel extraction, ligation and transformation of pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September1|Wednesday, September 1]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September2|Thursday, September 2]]===<br />
* Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September3|Friday, September 3]]===<br />
* Colony PCR of ''E. coli'' transformed on previous day<br />
* PCR amplification of 1-3A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September4|Saturday, September 4]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
* Estimation of the amount of 1-3A PCR product<br />
<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September6|Monday, September 6]]===<br />
*Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September7|Tuesday, September 7]]===<br />
*Concentration check of DNA used for ligation yesterday<br />
*Ethanol precipitation<br />
*Colony PCR of competent cells<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September8|Wednesday, September 8]]===<br />
*Colony PCR<br />
*Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3<br />
*PCR of GFP<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September9|Thursday, September 9]]===<br />
* 3 piece ligation of HSP, GFP and pSB1C3<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]===<br />
*3 piece ligation, continued<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September13|Monday, September 13]]===<br />
* AraC promoter purification<br />
* And follow up checks for quality<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September14|Tuesday, September 14]]===<br />
* Digestion and ligation of pSB1C3, araC Promoter and GFP<br />
* Ethanol precipitation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September15|Wednesday, September 15]]===<br />
*Observed results of yesterdays transformation<br />
**Transformation using heat shock went well<br />
**Electroporation transformation failed produce colonies<br />
*Did Colony PCR of yesterdays transformed colonies<br />
*Introduced colonies to L(+)Arabinose medium to check if it would show desired function<br />
**Check for results tomorrow<br />
<br />
===Thursday, September 16===<br />
*Observed results of overnight incubation<br />
**Fluorescence was visible when viewed by fluorescence microscope<br />
*Did experiment to see if fluorescence is affected by arabinose concentration<br />
*Scanned GFP intensity of broth containing colonies we isolated yesterday<br />
*Had a free time so amplified some parts for easy training constructs<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September17|Friday, September 17]]===<br />
*Construction of GFP marker for a part which will be secreted using T3SS<br />
*Ordered primers for construction for same part<br />
----<br />
<br />
===Monday, September 20===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]===<br />
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos<br />
<br />
===Wednesday, September 22===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]===<br />
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification<br />
*Colony PCR of AraC+RBS+pSB1A3<br />
*Electroporetion of BAC plasmid into DH5α MG1655<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September24|Friday, September 24]]===<br />
*Miniprep of Arac+RBS+pSB1A3<br />
*Follow quality check<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===<br />
* Culture of the BAC clones<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]===<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September29|Thursday, September 29]]===<br />
*Electrophoresis of Yesterday Sample<br />
*Colony PCR and cultivation of Arabinose promoter + RBS + GFP + double terminator <br />
*making competent cell of E.coli with SPI2<br />
*PCR of T3SS signal Again<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]===<br />
*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3<br />
*Transformation<br />
<br />
===Friday, October 1===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]===<br />
* Colony PCR<br />
* Preparation for Sequencing<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]===<br />
* Miniprep<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October6|Wednesday, October 6]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Saturday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October12|Tuesday, October 12]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October13|Wednesday, October 13]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October16|Saturday, October 16]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October17|Sunday, October 17]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October19|Tuesday, October 19]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October20|Wednesday, October 20]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October23|Saturday, October 23]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October24|Sunday, October 24]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October26|Tuesday, October 26]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October27|Wednesday, October 27]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October30|Saturday, October 30]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October31|Sunday, October 31]]===</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T17:32:44Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making of competent cell of E.coli with SPI2 BAC vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
We used special primers in this PCR which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be:<br />
<br />
<br><br />
EcoRI+XbaI+RBS+SlrP+SpeI+PstI<br />
<br><br><br><br />
Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be:<br />
<br><br><br />
EcoRI+XbaI+NLSx3GFP+doubleterminato+SpeI+PstI<br />
<br><br><br />
PCR mix<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><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 />
*PCRed according to the table below.98C and 68C for 45 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction.<br />
Stored at -20C.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T17:28:34Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<br />
<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|center|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|center|thumb|Electrophoresis of colony PCR sample]]<br />
<br />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.<br />
<br />
<br />
= PCR of T3SS signal again =<br />
<br />
*We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.<br />
<br />
PCR mix<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|7 uL<br />
|-<br />
|EX-F<br />
|1.5 uL<br />
|-<br />
|PS-R <br />
|1.5 uL<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div><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 />
*PCRed according to the table below.98C and 68C for 45 cycles in PCR of KOD. 94C 30 sec and 68C for 40 cycle in PCR of Quick Taq.<br />
<br />
<br><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
*Electrophoresed the samples and pSB1C3 and pSB1T3 which were amplified before.A band of T3SS signal amplified by Quick Taq was observed, but by KOD plus neo wasn't done.Estimated a concentration of T3SS signal and plasmid.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T17:24:41Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|center|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<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 />
<br />
<br />
<br />
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|center|thumb|Electrophoresis of colony PCR sample]]<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 />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.<br />
<br />
= PCR of T3SS signal again =<br />
<br />
*We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.<br />
<br />
PCR mix<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|7 uL<br />
|-<br />
|EX-F<br />
|1.5 uL<br />
|-<br />
|PS-R <br />
|1.5 uL<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div><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 />
<br />
<br />
*PCRed according to the table below.98C and 68C for 45 cycles in PCR of KOD. 94C 30 sec and 68C for 40 cycle in PCR of Quick Taq.<br />
<br />
<br><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
</div><br />
<br />
*Electrophoresed the samples and pSB1C3 and pSB1T3 which were amplified before.A band of T3SS signal amplified by Quick Taq was observed, but by KOD plus neo wasn't done.Estimated a concentration of T3SS signal and plasmid.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T17:13:46Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<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 />
<br />
<br />
<br />
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|right|thumb|Electrophoresis of colony PCR sample]]<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 />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.<br />
<br />
= PCR of T3SS signal again =<br />
<br />
*We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.<br />
<br />
PCR mix<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|7 uL<br />
|-<br />
|EX-F<br />
|1.5 uL<br />
|-<br />
|PS-R <br />
|1.5 uL<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
<br />
*PCRed according to the table below.98C and 68C for 45 cycles in PCR of KOD. 94C 30 sec and 68C for 40 cycle in PCR of Quick Taq.<br />
<br />
<br><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T17:05:21Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
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<br />
<br />
<br />
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|right|thumb|Electrophoresis of colony PCR sample]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
<br />
<br />
<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.<br />
<br />
= PCR of T3SS signal again =<br />
<br />
*We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.<br />
<br />
PCR mix<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|7 uL<br />
|-<br />
|EX-F<br />
|1.5 uL<br />
|-<br />
|PS-R <br />
|1.5 uL<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T17:04:15Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making of competent cell of E.coli with SPI2 BAC vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
We used special primers in this PCR which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be:<br />
<br />
<br><br />
EcoRI+XbaI+RBS+SlrP+SpeI+PstI<br />
<br><br><br><br />
Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be:<br />
<br><br><br />
EcoRI+XbaI+NLSx3GFP+doubleterminato+SpeI+PstI<br />
<br><br><br />
PCR mix<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
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<br />
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<br />
<br />
<br />
<br />
<br />
<br />
<br />
*PCRed according to the table below.98C and 68C for 45 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction.Stored at -20C.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T16:58:45Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<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 />
<br />
<br />
<br />
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|right|thumb|Electrophoresis of colony PCR sample]]<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 />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.<br />
<br />
= PCR of T3SS signal again =<br />
<br />
*We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.<br />
<br />
PCR mix<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;">{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|7 uL<br />
|-<br />
|EX-F<br />
|1.5 uL<br />
|-<br />
|PS-R <br />
|1.5 uL<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T16:56:18Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<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 />
<br />
<br />
<br />
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|right|thumb|Electrophoresis of colony PCR sample]]<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 />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.<br />
<br />
= PCR of T3SS signal again =<br />
<br />
*We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.<br />
<br />
PCR mix<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;">{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|7 uL<br />
|-<br />
|EX-F<br />
|1.5 uL<br />
|-<br />
|PS-R <br />
|1.5 uL<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T16:54:13Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<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 />
<br />
<br />
<br />
= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|right|thumb|Electrophoresis of colony PCR sample]]<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 />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.<br />
<br />
= PCR of T3SS signal again =<br />
<br />
*We thought colony PCR can't use KOD plus neo.We did a PCR with KOD plus neo again,and also did with Quick Taq.<br />
<br />
PCR mix<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left;">{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|7 uL<br />
|-<br />
|EX-F<br />
|1.5 uL<br />
|-<br />
|PS-R <br />
|1.5 uL<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/NotebookTest
Team:HokkaidoU Japan/NotebookTest
2010-10-27T16:39:56Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
__NOTOC__<br />
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.calendar a {<br />
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==Calendar==<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|July<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
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|2<br />
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|6<br />
|7<br />
|8<br />
|9<br />
|style="color:blue;"|10<br />
|-<br />
|style="color:red;"|11<br />
|[[#Monday, July 12|12]]<br />
|13<br />
|14<br />
|15<br />
|16<br />
|style="color:blue;"|17<br />
|-<br />
|style="color:red;"|18<br />
|19<br />
|20<br />
|[[#Wednesday, July 21|21]]<br />
|22<br />
|23<br />
|style="color:blue;"|24<br />
|-<br />
|style="color:red;"|25<br />
|[[#Monday, July 26|26]]<br />
|[[#Tuesday, July 27|27]]<br />
|28<br />
|29<br />
|30<br />
|style="color:blue;"|31<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|August<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|1<br />
|[[#Monday, August 2|2]]<br />
|3<br />
|4<br />
|5<br />
|6<br />
|style="color:blue;"|7<br />
|-<br />
|style="color:red;"|8<br />
|[[#Monday, August 9|9]]<br />
|[[#Tuesday, August 10|10]]<br />
|[[#Wednesday, August 11|11]]<br />
|[[#Thursday, August 12|12]]<br />
|[[#Friday, August 13|13]]<br />
|style="color:blue;"|14<br />
|-<br />
|style="color:red;"|15<br />
|[[#Monday, August 16|16]]<br />
|[[#Tuesday, August 17|17]]<br />
|[[#Wednesday, August 18|18]]<br />
|[[#Thursday, August 19|19]]<br />
|[[#Friday, August 20|20]]<br />
|style="color:blue;"|21<br />
|-<br />
|style="color:red;"|22<br />
|[[#Monday, August 23|23]]<br />
|[[#Tuesday, August 24|24]]<br />
|[[#Wednesday, August 25|25]]<br />
|[[#Thursday, August 26|26]]<br />
|[[#Friday, August 27|27]]<br />
|style="color:blue;"|28<br />
|-<br />
|style="color:red;"|29<br />
|[[#Monday, August 30|30]]<br />
|[[#Tuesday, August 31|31]]<br />
|<br />
|<br />
| <br />
|style="color:blue;"|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|September<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|[[#Wednesday, September 1|1]]<br />
|[[#Thursday, September 2|2]]<br />
|[[#Friday, September 3|3]]<br />
|style="color:blue;"|[[#Saturday, September 4|4]]<br />
|-<br />
|style="color:red;"|5<br />
|[[#Monday, September 6|6]]<br />
|[[#Tuesday, September 7|7]]<br />
|[[#Wednesday, September 8|8]]<br />
|[[#Thursday, September 9|9]]<br />
|[[#Friday, September 10|10]]<br />
|style="color:blue;"|11<br />
|-<br />
|style="color:red;"|12<br />
|[[#Monday, September 13|13]]<br />
|[[#Tuesday, September 14|14]]<br />
|[[#Wednesday, September 15|15]]<br />
|[[#Thursday, September 16|16]]<br />
|[[#Friday, September 17|17]]<br />
|style="color:blue;"|18<br />
|-<br />
|style="color:red;"|19<br />
|20<br />
|[[#Tuesday, September 21|21]]<br />
|[[#Wednesday, September 22|22]]<br />
|[[#Thursday, September 23|23]]<br />
|[[#Friday, September 24|24]]<br />
|style="color:blue;"|25<br />
|-<br />
|style="color:red;"|26<br />
|[[#Monday, September 27|27]]<br />
|[[#Tuesday, September 28|28]]<br />
|[[#Wednesday, September 29|29]]<br />
|[[#Thursday, September 30|30]]<br />
|<br />
|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|October<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|1<br />
|style="color:blue;"|[[#Saturday, October 2|2]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 3|3]]<br />
|[[#Monday, October 4|4]]<br />
|[[#Tuesday, October 5|5]]<br />
|[[#Wednesday, October 6|6]]<br />
|[[#Thursday, October 7|7]]<br />
|[[#Friday, October 8|8]]<br />
|style="color:blue;"|[[#Saturday, October 9|9]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 10|10]]<br />
|[[#Monday, October 11|11]]<br />
|[[#Tuesday, October 12|12]]<br />
|[[#Wednesday, October 13|13]]<br />
|[[#Thursday, October 14|14]]<br />
|[[#Friday, October 15|15]]<br />
|style="color:blue;"|[[#Saturday, October 16|16]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 17|17]]<br />
|[[#Monday, October 18|18]]<br />
|[[#Tuesday, October 19|19]]<br />
|[[#Wednesday, October 20|20]]<br />
|[[#Thursday, October 21|21]]<br />
|[[#Friday, October 22|22]]<br />
|style="color:blue;"|[[#Saturday, October 23|23]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 24|24]]<br />
|[[#Monday, October 25|25]]<br />
|[[#Tuesday, October 26|26]]<br />
|[[#Wednesday, October 27|27]]<br />
|[[#Thursday, October 28|28]]<br />
|[[#Friday, October 29|29]]<br />
|style="color:blue;"|[[#Saturday, October 30|30]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 31|31]]<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:blue;"|<br />
|}<br />
<div style="clear:both"></div><br />
<br />
==Abstract==<br />
===Monday, July 12===<br />
* Our first experiment, transformation of BioBrick devices.<br />
----<br />
<br />
===Wednesday, July 21===<br />
* Preparation of competent cells<br />
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12<br />
<br />
===Monday, July 26===<br />
* Cultivation of transformed ''E. coli'' for the next day's miniprep<br />
<br />
===Tuesday, July 27===<br />
* Miniprep (Alkaline SDS method)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August2|Monday, August 2]]===<br />
* Restriction enzyme digestion of plasmids which had been purified by miniprep<br />
* Electrophoresis assay<br />
<br />
===Tuesday, August 3===<br />
===Wednesday, August 4===<br />
===Thursday, August 5===<br />
===Friday, August 6===<br />
----<br />
===Monday, August 9===<br />
* Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive ''E. coli and heat-sensitive ''E. coli<br />
* These are preliminary experiments before starting the main project<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August10|Tuesday, August 10]]===<br />
* PCR amplification of the other parts (BBa_F1610 "2-24G", BBa_B0034 "1-2M", BBa_B0015 "1-23L" and BBa_K098995 "3-1E") and electrophoresis to confirm<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August11|Wednesday, August 11]]===<br />
* Preparation for the next day's miniprep<br />
* Estimation of enzyme activity<br />
* Preparation of the glycerol stocks<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August12|Thursday, August 12]]===<br />
* Miniprep (1-18F, 2-21H and 2-11P)<br />
* Electrophoresis of the DNA which had been amplified via PCR and miniprep<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August13|Friday, August 13]]===<br />
* PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August16|Monday, August 16]]===<br />
* Measurement of restriction enzyme activity<br />
* Restriction enzyme digestion of the parts(3-1E, 1-2M, 1-18F and 1-23L) and the vector(pSB1C3)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August17|Tuesday, August 17]]===<br />
* Purification of the DNA solutions via gel extraction<br />
* Preparation of agar medium containing 35 ug/mL chloramphenicol<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August18|Wednesday, August 18]]===<br />
* Digestion and gel extraction of 1-2M (retry)<br />
* 3 piece ligation of 1-18F, 1-23L and pSB1C3<br />
* Transformation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August19|Thursday, August 19]]===<br />
* 3 piece ligation of 3-1E (heat sensor), 1-2M (ribosome binding site) and pSB1C3 (vector)<br />
* Ligation that uses only vector pSB1C3 to estimate its efficiency<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August20|Friday, August 20]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August23|Monday, August 23]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)<br />
* PCR amplification of BioBrick parts using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August24|Tuesday, August 24]]===<br />
* Electrophoresis of PCR products that had been amplified using new primers<br />
* Examination of two ligation kits<br />
* Electrophoresis assay of miscellaneous DNA solutions<br />
* Transformation of 1-3A (RFP reporter with chloramphenicol resistance) to evaluate the medium<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August25|Wednesday, August 25]]===<br />
* Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium<br />
* Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation<br />
* Digestion of PCR products that had been amplified by using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August26|Thursday, August 26]]===<br />
* Electriphoresis to check whether ligation had been successful<br />
* Digestion of PCR products that had been amplified using new primers (with reconstruction of reaction system)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August27|Friday, August 27]]===<br />
* Ligation of 1-1A (RFP reporter device) into pSB1C3 and its transformation<br />
* Retry of 3 piece ligation which was done on August 19th and 20th<br />
* Ligation of vectors to each other<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August30|Monday, August 30]]===<br />
* Measurement of restriction enzyme activity using highly purified pUC119<br />
* Digestion of PCR products that had been amplified using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August31|Tuesday, August 31]]===<br />
* Gel extraction, ligation and transformation of pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September1|Wednesday, September 1]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September2|Thursday, September 2]]===<br />
* Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September3|Friday, September 3]]===<br />
* Colony PCR of ''E. coli'' transformed on previous day<br />
* PCR amplification of 1-3A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September4|Saturday, September 4]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
* Estimation of the amount of 1-3A PCR product<br />
<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September6|Monday, September 6]]===<br />
*Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September7|Tuesday, September 7]]===<br />
*Concentration check of DNA used for ligation yesterday<br />
*Ethanol precipitation<br />
*Colony PCR of competent cells<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September8|Wednesday, September 8]]===<br />
*Colony PCR<br />
*Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3<br />
*PCR of GFP<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September9|Thursday, September 9]]===<br />
* 3 piece ligation of HSP, GFP and pSB1C3<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]===<br />
*3 piece ligation, continued<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September13|Monday, September 13]]===<br />
* AraC promoter purification<br />
* And follow up checks for quality<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September14|Tuesday, September 14]]===<br />
* Digestion and ligation of pSB1C3, araC Promoter and GFP<br />
* Ethanol precipitation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September15|Wednesday, September 15]]===<br />
*Observed results of yesterdays transformation<br />
**Transformation using heat shock went well<br />
**Electroporation transformation failed produce colonies<br />
*Did Colony PCR of yesterdays transformed colonies<br />
*Introduced colonies to L(+)Arabinose medium to check if it would show desired function<br />
**Check for results tomorrow<br />
<br />
===Thursday, September 16===<br />
*Observed results of overnight incubation<br />
**Fluorescence was visible when viewed by fluorescence microscope<br />
*Did experiment to see if fluorescence is affected by arabinose concentration<br />
*Scanned GFP intensity of broth containing colonies we isolated yesterday<br />
*Had a free time so amplified some parts for easy training constructs<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September17|Friday, September 17]]===<br />
*Construction of GFP marker for a part which will be secreted using T3SS<br />
*Ordered primers for construction for same part<br />
----<br />
<br />
===Monday, September 20===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]===<br />
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos<br />
<br />
===Wednesday, September 22===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]===<br />
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification<br />
*Colony PCR of AraC+RBS+pSB1A3<br />
*Electroporetion of BAC plasmid into DH5α MG1655<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September24|Friday, September 24]]===<br />
*Miniprep of Arac+RBS+pSB1A3<br />
*Follow quality check<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===<br />
* Culture of the BAC clones<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]===<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September29|Wednesday, September 29]]===<br />
*Electrophoresis of T3SS signal and GFP + double terminator<br />
*Colony PCR and cultivation of arabinose promoter + RBS + GFP + double terminator<br />
*making competent cell of E.coli with SPI2<br />
*PCR of T3SS signal Again<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]===<br />
*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3<br />
*Transformation<br />
<br />
===Friday, October 1===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]===<br />
* Colony PCR<br />
* Preparation for Sequencing<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]===<br />
* Miniprep<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October6|Wednesday, October 6]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Saturday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October12|Tuesday, October 12]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October13|Wednesday, October 13]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October16|Saturday, October 16]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October17|Sunday, October 17]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October19|Tuesday, October 19]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October20|Wednesday, October 20]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October23|Saturday, October 23]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October24|Sunday, October 24]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October26|Tuesday, October 26]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October27|Wednesday, October 27]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October30|Saturday, October 30]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October31|Sunday, October 31]]===</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T16:32:28Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<br />
<br />
<br />
<br />
<br />
<br />
<br />
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= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
[[Image:HokkaidoU Japan 20100929b.jpg|200px|left|thumb|Electrophoresis of colony PCR sample]]<br />
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<br />
= Cultivation of colony =<br />
<br />
*cultivated the colonies which were used for PCR.Each Samples were cultured in LB contained Ampicillin and 20% Arabinose or contained only Ampicillin.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T16:21:59Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<br />
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= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
[[Image:HokkaidoU Japan 20100929b.JPG|200px|left|thumb|Electrophoresis of colony PCR sample]]<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
<br />
*</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September24
Team:HokkaidoU Japan/Notebook/September24
2010-10-27T16:19:51Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September23|September 23]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div></div><br />
<br />
*Miniprep of Arac+RBS+pSB1A3 <br />
*Follow quality check<br />
<br />
= Miniprep of Arac+RBS+pSB1A3 =<br />
<br />
#Transfered E.coli solution to 1.5 mL tubes, 1 mL each<br />
#Centrifuged at 4C, 15000 rpm for 1 min<br />
#Discarded the supernatant <br />
#Suspended on 125 uL of Buffer P1 each<br />
#Added 175 uL Buffer N3 each mixed by inversion<br />
#Centrifuged at 4C, 13000 rpm for 10 min<br />
#Transfered the supernatant to filtration column<br />
#Centrifuged at 4C, 13000 rpm for 1 min<br />
#Discarded the flow-through<br />
#added 500 uL of Buffer PB to filtration column<br />
#Centrifuged at 4C, 13000 rpm for 1 min<br />
#Discarded the flow-through centrifuged for 1min to remove remaining buffer<br />
#Transfered filtration column to a new 1.5 ml tube<br />
#Resuspended on 50 ul of TE and incubated at RT for 1min<br />
#Centrifuged at 4C, 13000 rpm for 1 min<br />
<br />
<br />
== Electrophoresis of minipreped samples ==<br />
#Mixed 1 uL of sample with loading buffer 1 uL<br />
#Added 6 uL of λ/Hind Ⅲ EcoR Marker<br />
#Electrophoresed<br />
<br />
<br />
[[Image:HokkaidoU Japan 20100924a.JPG|200px|left|thumb|Electrophoresis of minipreped samples]]<br />
<br />
Compared to marker plasmid is about 7000 bp long<br />
<br />
Arabinose Promoter+RBS+pSB1A3 is 3455 bp long, so it might be tandem<br />
<br />
From the picture estimate of concentration was about 30 ng/uL<br />
<br />
<br><br><br><br><br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
= Digestion of plasmid and GFP+double terminator =<br />
Digestion mix According to the table below<br />
<br />
{|style="text-align: center ;float:left;" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|DW<br />
|5.6 uL<br />
|-<br />
|10x H Buffer<br />
|1 uL<br />
|-<br />
|0.1% BSA<br />
|1 uL<br />
|-<br />
|Spe I<br />
|0.2 uL<br />
|-<br />
|Pst I<br />
|0.2 uL<br />
|-<br />
|plasmid<br />
|2 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''10 uL'''<br />
|}<br />
{|style="text-align: center; float:left;" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|DW<br />
|34 uL<br />
|-<br />
|10x M Buffer<br />
|5 uL<br />
|-<br />
|0.1% BSA<br />
|5 uL<br />
|-<br />
|Xba I<br />
|4 uL<br />
|-<br />
|Pst I<br />
|0.4 uL<br />
|-<br />
|GFP+double terminator<br />
|1.6 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br />
#incubated at 37C for 60 min<br />
#purified samples with Mycrocon YM-10<br />
#sampleが500 uLになるようにTEを加え、カラムに移した。<br />
#centrifuged at 4C,14000 G for 1h<br />
#上澄みが30 uL前後残ったので、それを新しいcollection tubeに移し、カラムを逆さまにして挿入した。<br />
#centrifuged at 4C,1000 G for 3min<br />
#GFP+double terminatorが30 uL、plasmid25 uL回収できたので、各1/10量の3M CH3COONaを加えた。<br />
#100%EtOHを回収量の2.5倍量加え、voltexにかけた。<br />
#centrifuged at 4C,15000 rpm for 10min<br />
#上澄みを捨て、70%EtOHを500 uL加え、voltexにかけた。<br />
#centrifuged at 4C,15000rpm for 5min<br />
#上澄みを捨て、真空装置へ入れよく乾燥させた。<br />
#2 uLのTEで溶かした。<br />
#-20Cで凍結保存した。<br />
<br />
<br />
== Transformation of BAC Vector ==<br />
<br />
*Used DH5Alpha and MG1655 strains for electroporation<br />
*Plated at 19:15<br />
*Will be incubated for 18 h</div>
Sho N
http://2010.igem.org/File:HokkaidoU_Japan_20100929b.jpg
File:HokkaidoU Japan 20100929b.jpg
2010-10-27T16:13:57Z
<p>Sho N: colonyPCR</p>
<hr />
<div>colonyPCR</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T16:12:35Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<br />
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= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =<br />
*PCRed 5 colony.<br />
<br />
<br />
PCR mix <br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|Quick Taq<br />
|25 uL<br />
|-<br />
|EX-F<br />
|0.5 uL<br />
|-<br />
|PS-R <br />
|0.5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''26 uL'''<br />
|}<br />
<br />
*PCRed according to the table below.98C and 68C for 35 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|94C<br />
|30 sec<br />
|-<br />
|68C<br />
|90 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*erectrophoresed the samples.Marker is 10 uL λ/HindIII EcoRI.<br />
<br />
*</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T15:52:35Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
= Electrophoresis of Yesterday Samples =<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.<br />
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= Colony PCR of Arabinose Promoter + RBS + GFP + double terminator =</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T15:43:04Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making of competent cell of E.coli with SPI2 BAC vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
We used special primers in this PCR which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be:<br />
<br />
<br><br />
EcoRI+XbaI+RBS+SlrP+SpeI+PstI<br />
<br><br><br><br />
Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be:<br />
<br><br><br />
EcoRI+XbaI+NLSx3GFP+doubleterminato+SpeI+PstI<br />
<br><br><br />
PCR mix<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
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<br />
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<br />
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<br />
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<br />
<br />
<br />
*PCRed according to the table below.98C and 68C for 45 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction.<br />
Stored at -20C.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T15:42:40Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making of competent cell of E.coli with SPI2 BAC vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
We used special primers in this PCR which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be:<br />
<br />
<br><br />
EcoRI+XbaI+RBS+SlrP+SpeI+PstI<br />
<br><br><br><br />
Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be:<br />
<br><br><br />
EcoRI+XbaI+NLSx3GFP+doubleterminato+SpeI+PstI<br />
<br><br><br />
PCR mix<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
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<br />
<br />
<br />
<br />
<br />
*PCRed according to the table below.98C and 68C for 45 cycles. <br />
<br />
<br><br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction.<br />
Stored at -20C.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T15:41:16Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making of competent cell of E.coli with SPI2 BAC vector<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
We used special primers in this PCR which added EcoRI ,XbaI, RBS SpeI and PstI restriction sites to SlrP found BAC vector(B_STM02P01 SGSC401) to make a biobrick. So the finished part would be:<br />
<br />
<br><br />
EcoRI+XbaI+RBS+SlrP+SpeI+PstI<br />
<br><br><br><br />
Similarly,GFP+doubleterminator was amplified by primer which had a region of EcoRI and XbaI and of three NLSs. To make a part which would be:<br />
<br><br><br />
EcoRI+XbaI+NLSx3GFP+doubleterminato+SpeI+PstI<br />
<br><br><br />
PCR mix<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
*PCRed according to the table below.98C and 68C for 45 cycles. <br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*electrophoresed the two samples, but a band of T3SS signal didn't show. Performed PCR and electrophoresis again,but a band of SlrP was weak. Cutted the band out and did gel extraction.<br />
Stored at -20C.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T15:39:06Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two yesterday samples.T3SS signal wasn't observed.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/NotebookTest
Team:HokkaidoU Japan/NotebookTest
2010-10-27T15:36:09Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
__NOTOC__<br />
<html><br />
<style><br />
#main h3{<br />
font-size: 116%; <br />
font-weight:normal;<br />
text-decoration:none;<br />
}<br />
<br />
#main h3 a{<br />
font-size: 116%; <br />
font-weight:normal;<br />
text-decoration:none;<br />
}<br />
<br />
#main ul {<br />
font-family:cursive;<br />
}<br />
<br />
<br />
.calendar {<br />
background-color:#F1F0E7;<br />
text-align:center;<br />
border:1px solid #630;<br />
float:left;<br />
margin:0px 10px 0px 5px;<br />
width:180px;<br />
}<br />
<br />
.calendar a {<br />
display: block;<br />
font-weight: bold;<br />
text-decoration: none;<br />
}<br />
<br />
</style><br />
</html><br />
==Calendar==<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|July<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|<br />
|1<br />
|2<br />
|style="color:blue;"|3<br />
|-<br />
|style="color:red;"|4<br />
|5<br />
|6<br />
|7<br />
|8<br />
|9<br />
|style="color:blue;"|10<br />
|-<br />
|style="color:red;"|11<br />
|[[#Monday, July 12|12]]<br />
|13<br />
|14<br />
|15<br />
|16<br />
|style="color:blue;"|17<br />
|-<br />
|style="color:red;"|18<br />
|19<br />
|20<br />
|[[#Wednesday, July 21|21]]<br />
|22<br />
|23<br />
|style="color:blue;"|24<br />
|-<br />
|style="color:red;"|25<br />
|[[#Monday, July 26|26]]<br />
|[[#Tuesday, July 27|27]]<br />
|28<br />
|29<br />
|30<br />
|style="color:blue;"|31<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|August<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|1<br />
|[[#Monday, August 2|2]]<br />
|3<br />
|4<br />
|5<br />
|6<br />
|style="color:blue;"|7<br />
|-<br />
|style="color:red;"|8<br />
|[[#Monday, August 9|9]]<br />
|[[#Tuesday, August 10|10]]<br />
|[[#Wednesday, August 11|11]]<br />
|[[#Thursday, August 12|12]]<br />
|[[#Friday, August 13|13]]<br />
|style="color:blue;"|14<br />
|-<br />
|style="color:red;"|15<br />
|[[#Monday, August 16|16]]<br />
|[[#Tuesday, August 17|17]]<br />
|[[#Wednesday, August 18|18]]<br />
|[[#Thursday, August 19|19]]<br />
|[[#Friday, August 20|20]]<br />
|style="color:blue;"|21<br />
|-<br />
|style="color:red;"|22<br />
|[[#Monday, August 23|23]]<br />
|[[#Tuesday, August 24|24]]<br />
|[[#Wednesday, August 25|25]]<br />
|[[#Thursday, August 26|26]]<br />
|[[#Friday, August 27|27]]<br />
|style="color:blue;"|28<br />
|-<br />
|style="color:red;"|29<br />
|[[#Monday, August 30|30]]<br />
|[[#Tuesday, August 31|31]]<br />
|<br />
|<br />
| <br />
|style="color:blue;"|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|September<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|[[#Wednesday, September 1|1]]<br />
|[[#Thursday, September 2|2]]<br />
|[[#Friday, September 3|3]]<br />
|style="color:blue;"|[[#Saturday, September 4|4]]<br />
|-<br />
|style="color:red;"|5<br />
|[[#Monday, September 6|6]]<br />
|[[#Tuesday, September 7|7]]<br />
|[[#Wednesday, September 8|8]]<br />
|[[#Thursday, September 9|9]]<br />
|[[#Friday, September 10|10]]<br />
|style="color:blue;"|11<br />
|-<br />
|style="color:red;"|12<br />
|[[#Monday, September 13|13]]<br />
|[[#Tuesday, September 14|14]]<br />
|[[#Wednesday, September 15|15]]<br />
|[[#Thursday, September 16|16]]<br />
|[[#Friday, September 17|17]]<br />
|style="color:blue;"|18<br />
|-<br />
|style="color:red;"|19<br />
|20<br />
|[[#Tuesday, September 21|21]]<br />
|[[#Wednesday, September 22|22]]<br />
|[[#Thursday, September 23|23]]<br />
|[[#Friday, September 24|24]]<br />
|style="color:blue;"|25<br />
|-<br />
|style="color:red;"|26<br />
|[[#Monday, September 27|27]]<br />
|[[#Tuesday, September 28|28]]<br />
|[[#Wednesday, September 29|29]]<br />
|[[#Thursday, September 30|30]]<br />
|<br />
|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|October<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|1<br />
|style="color:blue;"|[[#Saturday, October 2|2]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 3|3]]<br />
|[[#Monday, October 4|4]]<br />
|[[#Tuesday, October 5|5]]<br />
|[[#Wednesday, October 6|6]]<br />
|[[#Thursday, October 7|7]]<br />
|[[#Friday, October 8|8]]<br />
|style="color:blue;"|[[#Saturday, October 9|9]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 10|10]]<br />
|[[#Monday, October 11|11]]<br />
|[[#Tuesday, October 12|12]]<br />
|[[#Wednesday, October 13|13]]<br />
|[[#Thursday, October 14|14]]<br />
|[[#Friday, October 15|15]]<br />
|style="color:blue;"|[[#Saturday, October 16|16]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 17|17]]<br />
|[[#Monday, October 18|18]]<br />
|[[#Tuesday, October 19|19]]<br />
|[[#Wednesday, October 20|20]]<br />
|[[#Thursday, October 21|21]]<br />
|[[#Friday, October 22|22]]<br />
|style="color:blue;"|[[#Saturday, October 23|23]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 24|24]]<br />
|[[#Monday, October 25|25]]<br />
|[[#Tuesday, October 26|26]]<br />
|[[#Wednesday, October 27|27]]<br />
|[[#Thursday, October 28|28]]<br />
|[[#Friday, October 29|29]]<br />
|style="color:blue;"|[[#Saturday, October 30|30]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 31|31]]<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:blue;"|<br />
|}<br />
<div style="clear:both"></div><br />
<br />
==Abstract==<br />
===Monday, July 12===<br />
* Our first experiment, transformation of BioBrick devices.<br />
----<br />
<br />
===Wednesday, July 21===<br />
* Preparation of competent cells<br />
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12<br />
<br />
===Monday, July 26===<br />
* Cultivation of transformed ''E. coli'' for the next day's miniprep<br />
<br />
===Tuesday, July 27===<br />
* Miniprep (Alkaline SDS method)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August2|Monday, August 2]]===<br />
* Restriction enzyme digestion of plasmids which had been purified by miniprep<br />
* Electrophoresis assay<br />
<br />
===Tuesday, August 3===<br />
===Wednesday, August 4===<br />
===Thursday, August 5===<br />
===Friday, August 6===<br />
----<br />
===Monday, August 9===<br />
* Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive ''E. coli and heat-sensitive ''E. coli<br />
* These are preliminary experiments before starting the main project<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August10|Tuesday, August 10]]===<br />
* PCR amplification of the other parts (BBa_F1610 "2-24G", BBa_B0034 "1-2M", BBa_B0015 "1-23L" and BBa_K098995 "3-1E") and electrophoresis to confirm<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August11|Wednesday, August 11]]===<br />
* Preparation for the next day's miniprep<br />
* Estimation of enzyme activity<br />
* Preparation of the glycerol stocks<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August12|Thursday, August 12]]===<br />
* Miniprep (1-18F, 2-21H and 2-11P)<br />
* Electrophoresis of the DNA which had been amplified via PCR and miniprep<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August13|Friday, August 13]]===<br />
* PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August16|Monday, August 16]]===<br />
* Measurement of restriction enzyme activity<br />
* Restriction enzyme digestion of the parts(3-1E, 1-2M, 1-18F and 1-23L) and the vector(pSB1C3)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August17|Tuesday, August 17]]===<br />
* Purification of the DNA solutions via gel extraction<br />
* Preparation of agar medium containing 35 ug/mL chloramphenicol<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August18|Wednesday, August 18]]===<br />
* Digestion and gel extraction of 1-2M (retry)<br />
* 3 piece ligation of 1-18F, 1-23L and pSB1C3<br />
* Transformation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August19|Thursday, August 19]]===<br />
* 3 piece ligation of 3-1E (heat sensor), 1-2M (ribosome binding site) and pSB1C3 (vector)<br />
* Ligation that uses only vector pSB1C3 to estimate its efficiency<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August20|Friday, August 20]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August23|Monday, August 23]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)<br />
* PCR amplification of BioBrick parts using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August24|Tuesday, August 24]]===<br />
* Electrophoresis of PCR products that had been amplified using new primers<br />
* Examination of two ligation kits<br />
* Electrophoresis assay of miscellaneous DNA solutions<br />
* Transformation of 1-3A (RFP reporter with chloramphenicol resistance) to evaluate the medium<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August25|Wednesday, August 25]]===<br />
* Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium<br />
* Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation<br />
* Digestion of PCR products that had been amplified by using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August26|Thursday, August 26]]===<br />
* Electriphoresis to check whether ligation had been successful<br />
* Digestion of PCR products that had been amplified using new primers (with reconstruction of reaction system)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August27|Friday, August 27]]===<br />
* Ligation of 1-1A (RFP reporter device) into pSB1C3 and its transformation<br />
* Retry of 3 piece ligation which was done on August 19th and 20th<br />
* Ligation of vectors to each other<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August30|Monday, August 30]]===<br />
* Measurement of restriction enzyme activity using highly purified pUC119<br />
* Digestion of PCR products that had been amplified using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August31|Tuesday, August 31]]===<br />
* Gel extraction, ligation and transformation of pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September1|Wednesday, September 1]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September2|Thursday, September 2]]===<br />
* Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September3|Friday, September 3]]===<br />
* Colony PCR of ''E. coli'' transformed on previous day<br />
* PCR amplification of 1-3A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September4|Saturday, September 4]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
* Estimation of the amount of 1-3A PCR product<br />
<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September6|Monday, September 6]]===<br />
*Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September7|Tuesday, September 7]]===<br />
*Concentration check of DNA used for ligation yesterday<br />
*Ethanol precipitation<br />
*Colony PCR of competent cells<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September8|Wednesday, September 8]]===<br />
*Colony PCR<br />
*Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3<br />
*PCR of GFP<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September9|Thursday, September 9]]===<br />
* 3 piece ligation of HSP, GFP and pSB1C3<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]===<br />
*3 piece ligation, continued<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September13|Monday, September 13]]===<br />
* AraC promoter purification<br />
* And follow up checks for quality<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September14|Tuesday, September 14]]===<br />
* Digestion and ligation of pSB1C3, araC Promoter and GFP<br />
* Ethanol precipitation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September15|Wednesday, September 15]]===<br />
*Observed results of yesterdays transformation<br />
**Transformation using heat shock went well<br />
**Electroporation transformation failed produce colonies<br />
*Did Colony PCR of yesterdays transformed colonies<br />
*Introduced colonies to L(+)Arabinose medium to check if it would show desired function<br />
**Check for results tomorrow<br />
<br />
===Thursday, September 16===<br />
*Observed results of overnight incubation<br />
**Fluorescence was visible when viewed by fluorescence microscope<br />
*Did experiment to see if fluorescence is affected by arabinose concentration<br />
*Scanned GFP intensity of broth containing colonies we isolated yesterday<br />
*Had a free time so amplified some parts for easy training constructs<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September17|Friday, September 17]]===<br />
*Construction of GFP marker for a part which will be secreted using T3SS<br />
*Ordered primers for construction for same part<br />
----<br />
<br />
===Monday, September 20===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]===<br />
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos<br />
<br />
===Wednesday, September 22===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]===<br />
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification<br />
*Colony PCR of AraC+RBS+pSB1A3<br />
*Electroporetion of BAC plasmid into DH5α MG1655<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September24|Friday, September 24]]===<br />
*Miniprep of Arac+RBS+pSB1A3<br />
*Follow quality check<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===<br />
* Culture of the BAC clones<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]===<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September29|Wednesday, September 29]]===<br />
*Electrophoresis of T3SS signal and GFP + double terminator<br />
*Colony PCR and cultivation of arabinose promoter + RBS + GFP + double terminator<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]===<br />
*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3<br />
*Transformation<br />
<br />
===Friday, October 1===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]===<br />
* Colony PCR<br />
* Preparation for Sequencing<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]===<br />
* Miniprep<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October6|Wednesday, October 6]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Saturday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October12|Tuesday, October 12]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October13|Wednesday, October 13]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October16|Saturday, October 16]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October17|Sunday, October 17]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October19|Tuesday, October 19]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October20|Wednesday, October 20]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October23|Saturday, October 23]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October24|Sunday, October 24]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October26|Tuesday, October 26]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October27|Wednesday, October 27]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October30|Saturday, October 30]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October31|Sunday, October 31]]===</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/NotebookTest
Team:HokkaidoU Japan/NotebookTest
2010-10-27T15:34:24Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
__NOTOC__<br />
<html><br />
<style><br />
#main h3{<br />
font-size: 116%; <br />
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.calendar a {<br />
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font-weight: bold;<br />
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</style><br />
</html><br />
==Calendar==<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|July<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|<br />
|1<br />
|2<br />
|style="color:blue;"|3<br />
|-<br />
|style="color:red;"|4<br />
|5<br />
|6<br />
|7<br />
|8<br />
|9<br />
|style="color:blue;"|10<br />
|-<br />
|style="color:red;"|11<br />
|[[#Monday, July 12|12]]<br />
|13<br />
|14<br />
|15<br />
|16<br />
|style="color:blue;"|17<br />
|-<br />
|style="color:red;"|18<br />
|19<br />
|20<br />
|[[#Wednesday, July 21|21]]<br />
|22<br />
|23<br />
|style="color:blue;"|24<br />
|-<br />
|style="color:red;"|25<br />
|[[#Monday, July 26|26]]<br />
|[[#Tuesday, July 27|27]]<br />
|28<br />
|29<br />
|30<br />
|style="color:blue;"|31<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|August<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|1<br />
|[[#Monday, August 2|2]]<br />
|3<br />
|4<br />
|5<br />
|6<br />
|style="color:blue;"|7<br />
|-<br />
|style="color:red;"|8<br />
|[[#Monday, August 9|9]]<br />
|[[#Tuesday, August 10|10]]<br />
|[[#Wednesday, August 11|11]]<br />
|[[#Thursday, August 12|12]]<br />
|[[#Friday, August 13|13]]<br />
|style="color:blue;"|14<br />
|-<br />
|style="color:red;"|15<br />
|[[#Monday, August 16|16]]<br />
|[[#Tuesday, August 17|17]]<br />
|[[#Wednesday, August 18|18]]<br />
|[[#Thursday, August 19|19]]<br />
|[[#Friday, August 20|20]]<br />
|style="color:blue;"|21<br />
|-<br />
|style="color:red;"|22<br />
|[[#Monday, August 23|23]]<br />
|[[#Tuesday, August 24|24]]<br />
|[[#Wednesday, August 25|25]]<br />
|[[#Thursday, August 26|26]]<br />
|[[#Friday, August 27|27]]<br />
|style="color:blue;"|28<br />
|-<br />
|style="color:red;"|29<br />
|[[#Monday, August 30|30]]<br />
|[[#Tuesday, August 31|31]]<br />
|<br />
|<br />
| <br />
|style="color:blue;"|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|September<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|<br />
|<br />
|[[#Wednesday, September 1|1]]<br />
|[[#Thursday, September 2|2]]<br />
|[[#Friday, September 3|3]]<br />
|style="color:blue;"|[[#Saturday, September 4|4]]<br />
|-<br />
|style="color:red;"|5<br />
|[[#Monday, September 6|6]]<br />
|[[#Tuesday, September 7|7]]<br />
|[[#Wednesday, September 8|8]]<br />
|[[#Thursday, September 9|9]]<br />
|[[#Friday, September 10|10]]<br />
|style="color:blue;"|11<br />
|-<br />
|style="color:red;"|12<br />
|[[#Monday, September 13|13]]<br />
|[[#Tuesday, September 14|14]]<br />
|[[#Wednesday, September 15|15]]<br />
|[[#Thursday, September 16|16]]<br />
|[[#Friday, September 17|17]]<br />
|style="color:blue;"|18<br />
|-<br />
|style="color:red;"|19<br />
|20<br />
|[[#Tuesday, September 21|21]]<br />
|[[#Wednesday, September 22|22]]<br />
|[[#Thursday, September 23|23]]<br />
|[[#Friday, September 24|24]]<br />
|style="color:blue;"|25<br />
|-<br />
|style="color:red;"|26<br />
|[[#Monday, September 27|27]]<br />
|[[#Tuesday, September 28|28]]<br />
|[[#Wednesday, September 29|29]]<br />
|[[#Thursday, September 30|30]]<br />
|<br />
|<br />
|-<br />
|&nbsp;<br />
|}<br />
<br />
{|class="calendar"<br />
|-<br />
|colspan="7"|October<br />
|-<br />
!style="color:red;"|S<br />
!M<br />
!T<br />
!W<br />
!T<br />
!F<br />
!style="color:blue;"|S<br />
|-<br />
|style="color:red;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|1<br />
|style="color:blue;"|[[#Saturday, October 2|2]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 3|3]]<br />
|[[#Monday, October 4|4]]<br />
|[[#Tuesday, October 5|5]]<br />
|[[#Wednesday, October 6|6]]<br />
|[[#Thursday, October 7|7]]<br />
|[[#Friday, October 8|8]]<br />
|style="color:blue;"|[[#Saturday, October 9|9]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 10|10]]<br />
|[[#Monday, October 11|11]]<br />
|[[#Tuesday, October 12|12]]<br />
|[[#Wednesday, October 13|13]]<br />
|[[#Thursday, October 14|14]]<br />
|[[#Friday, October 15|15]]<br />
|style="color:blue;"|[[#Saturday, October 16|16]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 17|17]]<br />
|[[#Monday, October 18|18]]<br />
|[[#Tuesday, October 19|19]]<br />
|[[#Wednesday, October 20|20]]<br />
|[[#Thursday, October 21|21]]<br />
|[[#Friday, October 22|22]]<br />
|style="color:blue;"|[[#Saturday, October 23|23]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 24|24]]<br />
|[[#Monday, October 25|25]]<br />
|[[#Tuesday, October 26|26]]<br />
|[[#Wednesday, October 27|27]]<br />
|[[#Thursday, October 28|28]]<br />
|[[#Friday, October 29|29]]<br />
|style="color:blue;"|[[#Saturday, October 30|30]]<br />
|-<br />
|style="color:red;"|[[#Sunday, October 31|31]]<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:#555;"|<br />
|style="color:blue;"|<br />
|}<br />
<div style="clear:both"></div><br />
<br />
==Abstract==<br />
===Monday, July 12===<br />
* Our first experiment, transformation of BioBrick devices.<br />
----<br />
<br />
===Wednesday, July 21===<br />
* Preparation of competent cells<br />
* Single colony isolation of ''E. coli'' which had been transformed on Monday, July 12<br />
<br />
===Monday, July 26===<br />
* Cultivation of transformed ''E. coli'' for the next day's miniprep<br />
<br />
===Tuesday, July 27===<br />
* Miniprep (Alkaline SDS method)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August2|Monday, August 2]]===<br />
* Restriction enzyme digestion of plasmids which had been purified by miniprep<br />
* Electrophoresis assay<br />
<br />
===Tuesday, August 3===<br />
===Wednesday, August 4===<br />
===Thursday, August 5===<br />
===Friday, August 6===<br />
----<br />
===Monday, August 9===<br />
* Initial amplification of the BioBrick parts (BBa_I14032 "2-11P", BBa_F2621 "2-21H", BBa_K098995 "3-1E" and BBa_E1010 "1-18F") that are used in the construction of concentration-sensitive ''E. coli and heat-sensitive ''E. coli<br />
* These are preliminary experiments before starting the main project<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August10|Tuesday, August 10]]===<br />
* PCR amplification of the other parts (BBa_F1610 "2-24G", BBa_B0034 "1-2M", BBa_B0015 "1-23L" and BBa_K098995 "3-1E") and electrophoresis to confirm<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August11|Wednesday, August 11]]===<br />
* Preparation for the next day's miniprep<br />
* Estimation of enzyme activity<br />
* Preparation of the glycerol stocks<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August12|Thursday, August 12]]===<br />
* Miniprep (1-18F, 2-21H and 2-11P)<br />
* Electrophoresis of the DNA which had been amplified via PCR and miniprep<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August13|Friday, August 13]]===<br />
* PCR amplification of the DNA that we couldn't obtain via miniprep previous day(1-18F)<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August16|Monday, August 16]]===<br />
* Measurement of restriction enzyme activity<br />
* Restriction enzyme digestion of the parts(3-1E, 1-2M, 1-18F and 1-23L) and the vector(pSB1C3)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August17|Tuesday, August 17]]===<br />
* Purification of the DNA solutions via gel extraction<br />
* Preparation of agar medium containing 35 ug/mL chloramphenicol<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August18|Wednesday, August 18]]===<br />
* Digestion and gel extraction of 1-2M (retry)<br />
* 3 piece ligation of 1-18F, 1-23L and pSB1C3<br />
* Transformation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August19|Thursday, August 19]]===<br />
* 3 piece ligation of 3-1E (heat sensor), 1-2M (ribosome binding site) and pSB1C3 (vector)<br />
* Ligation that uses only vector pSB1C3 to estimate its efficiency<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August20|Friday, August 20]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August23|Monday, August 23]]===<br />
* PCR amplification of pSB1A3, pSB1C3 and pSB1T3 (with some changes in protocols)<br />
* PCR amplification of BioBrick parts using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August24|Tuesday, August 24]]===<br />
* Electrophoresis of PCR products that had been amplified using new primers<br />
* Examination of two ligation kits<br />
* Electrophoresis assay of miscellaneous DNA solutions<br />
* Transformation of 1-3A (RFP reporter with chloramphenicol resistance) to evaluate the medium<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August25|Wednesday, August 25]]===<br />
* Estimation of the amount of 1-3A DNA that could transform and grow cells on the chloramphenicol medium<br />
* Ethanol precipitation to condense the pSB1C3 DNA solution, and its digestion and ligation<br />
* Digestion of PCR products that had been amplified by using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August26|Thursday, August 26]]===<br />
* Electriphoresis to check whether ligation had been successful<br />
* Digestion of PCR products that had been amplified using new primers (with reconstruction of reaction system)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August27|Friday, August 27]]===<br />
* Ligation of 1-1A (RFP reporter device) into pSB1C3 and its transformation<br />
* Retry of 3 piece ligation which was done on August 19th and 20th<br />
* Ligation of vectors to each other<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August30|Monday, August 30]]===<br />
* Measurement of restriction enzyme activity using highly purified pUC119<br />
* Digestion of PCR products that had been amplified using new primers<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/August31|Tuesday, August 31]]===<br />
* Gel extraction, ligation and transformation of pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September1|Wednesday, September 1]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September2|Thursday, September 2]]===<br />
* Digestion, ligation and Transformation of RFP reporter with pSB1C3 or pUC119<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September3|Friday, September 3]]===<br />
* Colony PCR of ''E. coli'' transformed on previous day<br />
* PCR amplification of 1-3A (RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September4|Saturday, September 4]]===<br />
* PCR amplification of 1-5A (RFP reporter)<br />
* Estimation of the amount of 1-3A PCR product<br />
<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September6|Monday, September 6]]===<br />
*Ligation of pSB1C3 PCRed from 1-3A and 1-5A(RFP reporter)<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September7|Tuesday, September 7]]===<br />
*Concentration check of DNA used for ligation yesterday<br />
*Ethanol precipitation<br />
*Colony PCR of competent cells<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September8|Wednesday, September 8]]===<br />
*Colony PCR<br />
*Confirmation that pSB1C3 + RFP was not contaminated by template for pSB1C3<br />
*PCR of GFP<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September9|Thursday, September 9]]===<br />
* 3 piece ligation of HSP, GFP and pSB1C3<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September10|Friday, September 10]]===<br />
*3 piece ligation, continued<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September13|Monday, September 13]]===<br />
* AraC promoter purification<br />
* And follow up checks for quality<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September14|Tuesday, September 14]]===<br />
* Digestion and ligation of pSB1C3, araC Promoter and GFP<br />
* Ethanol precipitation<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September15|Wednesday, September 15]]===<br />
*Observed results of yesterdays transformation<br />
**Transformation using heat shock went well<br />
**Electroporation transformation failed produce colonies<br />
*Did Colony PCR of yesterdays transformed colonies<br />
*Introduced colonies to L(+)Arabinose medium to check if it would show desired function<br />
**Check for results tomorrow<br />
<br />
===Thursday, September 16===<br />
*Observed results of overnight incubation<br />
**Fluorescence was visible when viewed by fluorescence microscope<br />
*Did experiment to see if fluorescence is affected by arabinose concentration<br />
*Scanned GFP intensity of broth containing colonies we isolated yesterday<br />
*Had a free time so amplified some parts for easy training constructs<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September17|Friday, September 17]]===<br />
*Construction of GFP marker for a part which will be secreted using T3SS<br />
*Ordered primers for construction for same part<br />
----<br />
<br />
===Monday, September 20===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September21|Tuesday, September 21]]===<br />
*Annealing of RBS [http://partsregistry.org/wiki/index.php/Part:BBa_B0034 (BBa_B0034)] made from oligos<br />
<br />
===Wednesday, September 22===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September23|Thursday, September 23]]===<br />
*Amplifiable BAC plasmid ([http://partsregistry.org/Part:BBa_J61031 BBa_J61031]) purification<br />
*Colony PCR of AraC+RBS+pSB1A3<br />
*Electroporetion of BAC plasmid into DH5α MG1655<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September24|Friday, September 24]]===<br />
*Miniprep of Arac+RBS+pSB1A3<br />
*Follow quality check<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September27|Monday, September 27]]===<br />
* Culture of the BAC clones<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September28|Tuesday, September 28]]===<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
======<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September29|Wednesday, September 29]]===<br />
*Electrophoresis of T3SS signal and GFP + double terminator<br />
*Colony PCR and cultivation of arabinose promoter + RBS + GFP + double terminator<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/September30|Thursday, September 30]]===<br />
*Digestion and Ligation of Arabinose Promoter,T3SSsignal and pSB1T3<br />
*Transformation<br />
<br />
===Friday, October 1===<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October2|Saturday, October 2]]===<br />
* Colony PCR<br />
* Preparation for Sequencing<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October3|Sunday, October 3]]===<br />
* Miniprep<br />
----<br />
<br />
===[[Team:HokkaidoU_Japan/Notebook/October4|Monday, October 4]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October5|Tuesday, October 5]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October6|Wednesday, October 6]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October7|Thursday, October 7]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Friday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October8|Saturday, October 8]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October10|Sunday, October 10]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October11|Monday, October 11]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October12|Tuesday, October 12]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October13|Wednesday, October 13]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October14|Thursday, October 14]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October15|Friday, October 15]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October16|Saturday, October 16]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October17|Sunday, October 17]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October18|Monday, October 18]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October19|Tuesday, October 19]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October20|Wednesday, October 20]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October21|Thursday, October 21]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October22|Friday, October 22]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October23|Saturday, October 23]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October24|Sunday, October 24]]===<br />
----<br />
===[[Team:HokkaidoU_Japan/Notebook/October25|Monday, October 25]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October26|Tuesday, October 26]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October27|Wednesday, October 27]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October28|Thursday, October 28]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October29|Friday, October 29]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October30|Saturday, October 30]]===<br />
===[[Team:HokkaidoU_Japan/Notebook/October31|Sunday, October 31]]===</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T15:18:00Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
*We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><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 />
<br />
*PCRed according to the table below.98C and 68C were 45 cycles. <br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*electrophoresed the two sample, but a band of T3SS signal wasn't seen.PCRed and electrophoresed again,but a band of T3SS signal wasn't seen clearly.それらしい影を切り出してゲル抽出した。</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T14:17:50Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
*We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><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 />
<br />
*PCRed according to the table below.98C and 68C were 45 cycles. <br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
*electrophoresed the two sample, but a band of T3SS signal wasn't seen.PCRed and electrophoresed again,but a band of T3SS signal wasn't seen clearly.Cut the</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T14:17:13Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
*We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<br />
*electrophoresed the two sample, but a band of T3SS signal wasn't seen.PCRed and electrophoresed again,but a band of T3SS signal wasn't seen clearly.Cut the <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 />
*PCRed according to the table below.98C and 68C were 45 cycles. <br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T14:06:32Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
[[Image:HokkaidoU Japan 20100929a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two samples.T3SS signal wasn't observed.</div>
Sho N
http://2010.igem.org/File:HokkaidoU_Japan_20100929a.jpg
File:HokkaidoU Japan 20100929a.jpg
2010-10-27T14:05:34Z
<p>Sho N: GFP-double terminator(T3SS signal wasn't observed)</p>
<hr />
<div>GFP-double terminator(T3SS signal wasn't observed)</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September29
Team:HokkaidoU Japan/Notebook/September29
2010-10-27T14:04:17Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September28|September 28]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September30|September 30]]</div></div><br />
<br />
[[Image:HokkaidoU Japan 20100826a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two samples.T3SS signal wasn't observed.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T13:58:33Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
*We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><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 />
<br />
<br />
*PCRed according to the table below.98C and 68C were 45 cycles. <br />
{|style="text-align: center" class="protocol"<br />
!temp<br />
!time<br />
|-<br />
|94C<br />
|2 min<br />
|-<br />
|98C<br />
|10 sec<br />
|-<br />
|68C<br />
|60 sec<br />
|-<br />
|4C<br />
|hold<br />
|-<br />
|}<br />
<br />
[[Image:HokkaidoU Japan 20100826a.jpg|200px|right|thumb|Electrophoresis of concentrated GFP-double terminator(T3SS signal wasn't seen)]]<br />
<br />
<br />
*electrophoresed 6 uL Marker λ/HindIII EcoRI and 1 uL of two samples.T3SS signal wasn't observed.</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/August27
Team:HokkaidoU Japan/Notebook/August27
2010-10-27T13:41:28Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/August26|August 26]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/August30|August 30]]</div></div><br />
<br />
=Transfer [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] on pSB1A10 to pSB1C3=<br />
1.estimated concentrations of pSB1C3 and [[Team:HokkaidoU_Japan/Materials_And_Methods#BioBricks|1-1A]] which was amplified by miniprep before<br />
<br />
2.mixed solutions according to the table below<br />
<br />
<br />
<div style="float:left;"><br />
{|style="text-align: center;" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
|pSB1C3<br />
|5 uL<br />
|-<br />
|DW<br />
|75 uL<br />
|-<br />
|10x M Buffer<br />
|10 uL<br />
|-<br />
|1%BSA<br />
|10 uL<br />
|-<br />
|EcoRI<br />
|5 uL<br />
|-<br />
|PstI<br />
|5 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''100 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center;" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-1A<br />
|8 uL<br />
|-<br />
|DW<br />
|6 uL<br />
|-<br />
|10x M Buffer<br />
|2 uL<br />
|-<br />
|1%BSA<br />
|2 uL<br />
|-<br />
|EcoRI<br />
|1 uL<br />
|-<br />
|PstI<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''20 uL'''<br />
|}<br />
</div><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 />
3.incubated at 37C for 60min<br />
<br />
4.purified the two samples<br />
<br />
5.added 250 uL 100% EtOH to each samples<br />
<br />
6.centrifuged at 4C, 15000rpm for 10 min<br />
<br />
7.discarded supernatant<br />
<br />
8.put the samples into desiccator<br />
<br />
9.added 10 uL DW to pSB1C3 and 2 uL DW to 1-1A<br />
<br />
10.mixed 1 uL pSB1C3 and 2 uL 1-1A<br />
<br />
11.added 3 uL ligation solution and 0.5 uL T4 ligase<br />
<br />
12.transfered the sample to a tube of 500 uL<br />
<br />
13.incubated at 16C for 30 min<br />
<br />
->For not putting 3M CH3COONa into the solutions,we stopped operation up to this.<br />
<br />
<br />
=Preparation of 3 Piece Ligation=<br />
<br />
*Parts Information<br />
{|border="1" style="margin-left: 20px;" class="protocol"<br />
|-<br />
!Name<br />
!Biobrick Name<br />
!Well<br />
!Length<br />
|-<br />
| Heat Sensor<br />
| BBa_K098995<br />
| 3-1E<br />
| 937 bp<br />
|-<br />
| RBS<br />
| BBa_B0034<br />
| 1-2M<br />
| 12 bp<br />
|-<br />
| RFP<br />
| BBa_E1010<br />
| 1-18F<br />
| 681 bp<br />
|-<br />
| Double Terminator<br />
| BBa_0015<br />
| 1-23L<br />
| 129 bp<br />
|}<br />
<br />
<br />
1.estimated concentrations of Heat Sensor,RBS,RFP and Double Terminator<br />
<br />
2.mixed solutions according to the table below<br />
<br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 13.5 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 1%BSA<br />
| 5 uL<br />
|-<br />
| EcoRI<br />
| 3 uL<br />
|-<br />
| SpeI<br />
| 1 uL<br />
|-<br />
| Heat Sensor<br />
| 22.5 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|} <br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 34.9 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 1%BSA<br />
| 5 uL<br />
|-<br />
| XbaI<br />
| 1 uL<br />
|-<br />
| PstI<br />
| 2 uL<br />
|-<br />
| RBS<br />
| 34.9 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}<br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 25.5 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 1%BSA<br />
| 5 uL<br />
|-<br />
| EcoRI<br />
| 3 uL<br />
|-<br />
| SpeI<br />
| 1 uL<br />
|-<br />
| RFP<br />
| 10.5 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}<br />
{|style="text-align: center; margin-left:20px" class="protocol"<br />
|-<br />
!Reagent<br />
!Amount<br />
|-<br />
| DW<br />
| 34 uL<br />
|-<br />
| 10x M buffer<br />
| 5 uL<br />
|-<br />
| 1%BSA<br />
| 5 uL<br />
|-<br />
| XbaI<br />
| 1 uL<br />
|-<br />
| PstI<br />
| 2 uL<br />
|-<br />
| Double Terminator<br />
| 3 uL<br />
|-<br />
|style="border-top:1px solid #996;"|'''Total'''<br />
|style="border-top:1px solid #996;"|'''50 uL'''<br />
|}</div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T13:36:52Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
*We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3 Primer<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T13:36:22Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
*We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.<br />
<div style="float:left;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div></div>
Sho N
http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September28
Team:HokkaidoU Japan/Notebook/September28
2010-10-27T13:35:39Z
<p>Sho N: </p>
<hr />
<div>{{Template:HokkaidoU_Japan}}<br />
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September27|September 27]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September29|September 29]]</div></div><br />
<br />
*glycerol-stock of E.coli with salmonella's BAC library vector<br />
*making competent cell of E.coli with SPI2<br />
*plasmid & GFP-double terminator's Ligation & Transformation<br />
*PCR of E.coli with T3SSsignal and of GFP-double terminator<br />
<br />
= Ligation of plasmid and GFP-double terminator & Transformation =<br />
#added 2 uL TE into plasmid solvant and GFP-double terminator solvant <br />
#mixed the samples<br />
#added 5 uL Mighty mix<br />
#incubated at 16C for 30min<br />
#added the sample to 100 uL competent cell<br />
#incubated at 0C for 30min<br />
#heatshocked at 42C for 60sec<br />
#incubated at 0C for 5min<br />
#added sample to 400 uL LB<br />
#incubated at 37C for 2 hours<br />
#plated the sample on LBA medium<br />
#incubated at 37C<br />
<br />
= PCR of Parts =<br />
<br />
*We used special primers in this PCR.Plasmids of T3SS signal don't have a region of EcoRI and XbaI and of SpeI and PstI.EX-RBS primer has a region of EcoRI and XbeI and of RBS,SlrP3 primer has a region of SpeI and PatI.These primer can bind the start and end of T3SS signal,so T3SS signal can amplify with restriction site and RBS.Similarly,GFP+doubleterminator was amplified by NLS primer which has a region of EcoRI and XbaI and of three NLSs.<br />
<br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|colony solution<br />
|10 uL<br />
|-<br />
|DW<br />
|23 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|EX-RBS Primer<br />
|1.5 uL<br />
|-<br />
|SlrP3<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}<br />
</div><br />
<div style="float:left; margin-left:50px;"><br />
{|style="text-align: center" class="protocol"<br />
!Reagent<br />
!Amount<br />
|-<br />
|1-12K<br />
|1 uL<br />
|-<br />
|DW<br />
|32.5 uL<br />
|-<br />
|10x PCR Buffer<br />
|5 uL<br />
|-<br />
|5 mM 4dNTPs<br />
|5 uL<br />
|-<br />
|25 mM MgSO4<br />
|3 uL<br />
|-<br />
|NLS Primer<br />
|1.5 uL<br />
|-<br />
|PS-R<br />
|1.5 uL<br />
|-<br />
|KOD plus neo<br />
|1 uL<br />
|-<br />
|style="border-top:1px solid #996"|'''Total'''<br />
|style="border-top:1px solid #996"|'''50 uL'''<br />
|}</div>
Sho N