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http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_9
Team:HokkaidoU Japan/Notebook/plastic Week 9
2012-09-27T04:02:34Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===August 27th===<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 120827 phaC digestion sugama.jpg|thumb|digestion result]]<br />
[[image:HokaidoU2012 120827 RBS digestion sugama.jpg|thumb|digestion result]]<br />
====Digestion====<br />
BBa_K342001(PhaC) was digested by XbaI and PstI.<br />And BBa_B0034(RBS) was digested by SpeI and PstI (with three samples).<br />
PhaC (BBa_K342001)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
RBS (BBa_B0034)<br />
N0. 1<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (20.3 ng/ul)<br />
|14.3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2.5 ul<br />
|-<br />
|DW<br />
|0.2 ul<br />
|-<br />
|Total<br />
|25 ul<br />
|}<br />
<br />
<br />
N0. 2<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (15.6 ng/ul)<br />
|18.6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2.5 ul<br />
|-<br />
|DW<br />
|1.9 ul<br />
|-<br />
|Total<br />
|25 ul<br />
|}<br />
<br />
<br />
N0. 3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (16.9 ng/ul)<br />
|17.2 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2.5 ul<br />
|-<br />
|DW<br />
|3.3 ul<br />
|-<br />
|Total<br />
|25 ul<br />
|}<br />
<br />
====Electrophoresis====<br />
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.<br />
<br />
====Gel extraction====<br />
The digestion product was extracted. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===August 28th===<br />
<div class="hokkaidou-section"><br />
====Digestion====<br />
Digestion to divide PhaA and PhaB with XbaI and PstI.<br />I digested PhaC(BBa_K342001) with these restriction sites and also XhoI to divide pSB1C3 into pieces, on which PhaC is, that is because the length of pSB1C3 is nearly PhaC.<br />And we digested PhaC (BBa_K342001) and pSB1C3 by XbaI and SpeI.<br /><br /><br />
PhaA<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (125 ng/ul)<br />
|6.6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
PhaB<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (125 ng/ul)<br />
|4 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
PhaC(BBa_K342001)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (125 ng/ul)<br />
|10 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|XhoI<br />
|5.1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|0.9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
====Electrophoresis====<br />
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.<br />
<br />
====Gel extraction====<br />
Gel extraction for digestion product. Used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===August 29th===<br />
<div class="hokkaidou-section"><br />
====PHB polymer ethanolysis====<br />
We did ethanolysisãof PHB polymer for 4 hrs with sample 2~8.<br />
<br />
====Preparation for GC/MS====<br />
We did the preparation for GC/MS with sample 2~8.<br />
<br />
====PCR====<br />
We multiplied pSB1C3 by PCR.<br />
Used two different DNA polymerase, KOD-Plus-Neo and KAPA Taq.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|Suffix-EX<br />
|1<br />
|-<br />
|Prefix-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|74<br />
|2<br />
|-<br />
|4<br />
|98<br />
|10<br />
|-<br />
|5<br />
|72<br />
|120<br />
|-<br />
|6<br />
|98<br />
|10<br />
|-<br />
|7<br />
|70<br />
|120<br />
|-<br />
|8<br />
|98<br />
|10<br />
|-<br />
|9<br />
|68<br />
|120<br />
|-<br />
|10<br />
|68<br />
|420<br />
|-<br />
|11<br />
|4<br />
|HOLD<br />
|}<br />
Cycle1 : 2~3 x 5<br />
Cycle2 : 4~5 x 5<br />
Cycle3 : 6~7 x 5<br />
Cycle4 : 8~9 x 30<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|Suffix-EX (10 uM)<br />
|2<br />
|-<br />
|Prefix-PS (10 uM)<br />
|2<br />
|-<br />
|KAPA Taq<br />
|25<br />
|-<br />
|DW<br />
|20<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|63.7<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===August 30th===<br />
<div class="hokkaidou-section"><br />
====Ethanol precipitation====<br />
Diegested phaA by XbaI and SpeI and RBS by SpeI were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
PhaA and RBS, PhaB and RBS, PhaB and pSB1C3 were ligated each other.<br />And the DNA were transformed into bacteria.<br />
<br />
====Colony PCR====<br />
The length of PhaB on pSB1C3 was confirmed by colony PCR.<br />The result showed PhaB and pSB1C3 didn't ligate correctly. <br />
<br />
====Liquid Culture====<br />
Incubation of bacteria holds RBS (BBa_B0034) - PhaC (K342001) was started.<br />
<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===August 31st===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
We confirmed the length of the three constructs that transformed at August 30th.<br />The result showed RBS and PhaA were ligated correctly.<br />So the incubation was started.<br />
[[image:HokkaidoU 120831 RBS phaA coloP edit (2).jpg|thumb|center|450px]]<br />
<br />
====Plasmid extraction====<br />
Plasmid of RBS-PhaC were extracted.<br />And then we got 50ul DNA solution.<br />
<br />
====Liquid culture====<br />
Incubation of bacteria holds dT (BBa_B0015) was started.<br />
<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 1st===<br />
<div class="hokkaidou-section"><br />
<br />
====Plasmid extraction====<br />
Plasmids of RBS-PhaA and dT (BBa_B0015) were extracted.<br />And then we got 50ul DNA solution.<br />
[[image:HokkaidoU 120901 dT RBS-PhaA RBS-PhaC mini-prepç£ç© edit.jpg|thumb|center|600px|center|Fig. Extracted plasmids of dT and RBS-PhaA on pSB1A2]]<br /><br />
1: dT on pSB1AK3 (About 3.3kbp)<br /><br />
2 to 4: RBS-PhaA on pSB1A2 (About 3.2kbp)<br /><br />
5 to 7: RBS-PhaC on pSB1A2 (About 4.1kbp)<br />We thought sample 4 is not ideal plasmid and trashed it.<br />
<br />
====Digestion====<br />
RBS-PhaA was digested by XbaI and SpeI restriction site to ligate with RBS-PhaC digested by SpeI site.<br />
[[image:HokkaidoU 120901 RBS-PhaA RBS-PhaC digestion.jpg]]<br />
*1 and 2 is digested RBS-PhaA on pSB1A2.<br />
*Upper fragment is vector, pSB1A2.<br />
*Lower one is an objective fragment, RBS-PhaA (About 1.2kbp).<br />
*And PhaB and pSB1C3 were digested with XbaI and SpeI site.<br/>We decided to try ligation PhaB with pSB1C3 again.<br />
<br />
====Electrophoresis====<br />
We confirmed success of digestion by electrophoresis.<br/>The DNA was extracted from TBE gel and we got DNA solution.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 2nd===<br />
<div class="hokkaidou-section"><br />
<br />
====Ethanol precipitation====<br />
The digested DNAs, RBS-PhaA (No. 1), RBS-PhaA (No. 2), RBS-PhaC on pSB1A2, PhaB and pSB1C3 were concentrated by Ethanol precipitation.<br />
<br />
====Ligation====<br />
RBS-PhaA (No. 1 and No. 2) was ligated with RBS-PhaC on pSB1A2.<br/><br />
And PhaB was taken in pSB1C3.<br />
<br />
====Transformation====<br />
These ligated DNAs transformed into E. coli (strain: DH5&alpha;).<br/><br />
And then we spread fungus liquid added LB on LB plates include antibiotics.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_8
Team:HokkaidoU Japan/Notebook/plastic Week 8
2012-09-27T04:00:32Z
<p>Kwt: /* Plasmid extraction */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===August 20th===<br />
<div class="hokkaidou-section"><br />
====Digestion====<br />
We digested 3 samples.<br><br />
Digested of PhaC and pSB1C3 by XbaI and SpeI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution PhaC <br />
|12 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Digested of PhaA by XbaI site and SpeI site.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution PhaA<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
And digested PhaB by XbaI site and SpeI site.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution PhaB<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===August 21st===<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 120821PhaA PhaB PhaC digestion okamura.jpg|thumb|digestion result]]<br />
====Electrophoresis====<br />
We confirmed whether PhaC was digested correctly, and phaA and phaB were done PCR correctly by electrophoresis. <br />
<br />
====Gel extraction====<br />
We confirmed succession of digestion by electrophoresis, then DNA were extracted from TBE gel.<br />
And we used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===August 22nd===<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU 2012 120822PhaA PhaB pSB1C3 ETOH.jpg|thumb|Ethanol precipitation result]]<br />
====Ethanol precipitation====<br />
The DNA extracted at August 21st were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
We ligated phaA with pSB1C3 and phaB with pSB1C3.<br />
<br />
====Transformation====<br />
The ligated DNA were transformed into E. coli (strain:DH5&alpha;).<br />
E. coli solution was spread on LBC.<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 23rd===<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU 120823 PhaAcolop1 14.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU 120823PhaBcolop1 14.jpg|thumb|Colony PCR result]]<br />
<br />
====Colony PCR====<br />
We confirmed whether the ligation at August 22nd went well by colony PCR result.<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 24th===<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 120824 PhaA coloP.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120824 PhaB coloP.jpg|thumb|Colony PCR result]]<br />
<br />
====Colony PCR====<br />
We did colony PCR of PhaA and PhaB again.<br />
<br />
From the result, we thought that we failed to ligate PhaA with pSB1C3 and PhaB with pSB1C3. So we decided to ligated them again.<br />
<br />
====Ligation====<br />
We ligated phaA and phaB with pSB1C3.<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 25th===<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU120825 phaAcolonyPCR.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120825 phaBcolonyPCR.jpg|thumb|Colony PCR result]]<br />
<br />
====Colony PCR====<br />
We confirmed the length of PhaA on pSB1C3 and PhaB on pSB1C3 by colony PCR.<br><br />
The result showed only PhaA and pSB1C3 were ligated correctly.<br />
<br />
====Liquid culture====<br />
We started to incubate bacteria holds RBS (B0034).<br />
<br />
====Digestion====<br />
We digested PhaC(BBa_K342001) by XbaI and SpeI. The result shows that the digestion was succeeded.<br />
<br />
[[image:HokkaidoU2012 120825 phaCdigestion.jpg|thumb|digestion result]]<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 26th===<br />
<div class="hokkaidou-section"><br />
[[image:HokkaidoU2012 120826 RBS&amp;PhaA.jpg|thumb|plasmid extraction result]]<br />
====Plasmid extraction====<br />
The plasmid of RBS (BBa_B0034) were extracted.<br/>And then we got 50 ul DNA solution.<br />
<br />
====Digestion====<br />
RBS (BBa_B0034) was digested with SpeI and PstI restriction sites.<br/>And also PhaC (BBa_K342001) was digested by XbaI and PstI.<br />
<br />
====Liquid culture====<br />
We started to incubate.<br />
<br style="line-height: 0; clear: both;" /><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_5
Team:HokkaidoU Japan/Notebook/plastic Week 5
2012-09-27T03:58:01Z
<p>Kwt: /* PHB production test */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===August 2nd===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
We transformed pGEM into E.coli (strain:BL21). This transformation is the '''bioplastic production test in BL21'''.<br />
#Added 1 ul of plasmid DNA solution to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto LBA dish and spread.<br />
#Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBA dish then spread. <br />
#Incubated the plates at 37C for 15 hrs 30 min.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 3rd===<br />
<div class="hokkaidou-section"><br />
====Liquid culture====<br />
Liquid culture of transformed BL21(DE3)<br />
#Added 2 ml of LBA into culture tubes.<br />
#Resuspended 2 colonies.<br />
#Incubated the tubes at 37C for 18 hrs.<br />
<br />
After incubating, we tried to product PHB in BL21(DE3)<br />
<br />
====PHB production test====<br />
We prepared those three medium tubes and incubated them for 48 hrs.<br />
#700 ml LB + 0.7 ul Amp<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose + 1.4 ul Sodium pantothenate<br />
*BL21(DE3) = No. 1~3 incubated<br />
*JM109 = No. 2 only incubated (control)<br />
<br />
Today the requested parts to HQ were sent! Parts were sent as agar stabs. We note here how to use these agar stabs.<br />
Using agar stab(http://partsregistry.org/Help:Requesting_Parts)<br />
We send out our part requests as agar stabs. The shelf life of these are short, so it is usually best to plate from the stab as soon as possible.<br /><br />
The agar should already have a hole from when it was stabbed. With an inoculating loop dipped into the stab, you can plate directly onto a petri dish with the appropriate antibiotic.<br />
#Incubate the dish overnight at 37C (14-16 hrs)<br />
#Pick a single colony to start up a culture<br />
#Extract plasmid DNA<br />
#Use the part! <br />
<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_5
Team:HokkaidoU Japan/Notebook/plastic Week 5
2012-09-27T03:57:25Z
<p>Kwt: /* Liquid culture */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===August 2nd===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
We transformed pGEM into E.coli (strain:BL21). This transformation is the '''bioplastic production test in BL21'''.<br />
#Added 1 ul of plasmid DNA solution to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto LBA dish and spread.<br />
#Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBA dish then spread. <br />
#Incubated the plates at 37C for 15 hrs 30 min.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 3rd===<br />
<div class="hokkaidou-section"><br />
====Liquid culture====<br />
Liquid culture of transformed BL21(DE3)<br />
#Added 2 ml of LBA into culture tubes.<br />
#Resuspended 2 colonies.<br />
#Incubated the tubes at 37C for 18 hrs.<br />
<br />
After incubating, we tried to product PHB in BL21(DE3)<br />
<br />
====PHB production test====<br />
We prepared those three medium tubes and incubated them for 48 hrs.<br />
#700 ml LB + 0.7 ul Amp<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose + 1.4 ul Sodium pantothenate<br />
*BL21(DE3) = No. 1~3 incubated<br />
*JM109 = No. 2 only incubated (control)<br />
<br />
Today the requested parts to HQ were sent! Parts were sent as agar stabs. We note here how to use these agar stabs.<br />
Using agar stab(http://partsregistry.org/Help:Requesting_Parts)<br />
We send out our part requests as agar stabs. The shelf life of these are short, so it is usually best to plate from the stab as soon as possible.<br /><br />
The agar should already have a hole from when it was stabbed. With an inoculating loop dipped into the stab, you can plate directly onto a petri dish with the appropriate antibiotic.<br />
#Incubate the dish overnight at 37C (14-16hrs)<br />
#Pick a single colony to start up a culture<br />
#Extract plasmid DNA<br />
#Use the part! <br />
<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_5
Team:HokkaidoU Japan/Notebook/plastic Week 5
2012-09-27T03:56:58Z
<p>Kwt: /* PHB production test */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===August 2nd===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
We transformed pGEM into E.coli (strain:BL21). This transformation is the '''bioplastic production test in BL21'''.<br />
#Added 1 ul of plasmid DNA solution to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto LBA dish and spread.<br />
#Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBA dish then spread. <br />
#Incubated the plates at 37C for 15 hrs 30 min.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 3rd===<br />
<div class="hokkaidou-section"><br />
====Liquid culture====<br />
Liquid culture of transformed BL21(DE3)<br />
#Added 2 ml of LBA into culture tubes.<br />
#Resuspended 2 colonies.<br />
#Incubated the tubes at 37C for OOhrs.<br />
<br />
After incubating, we tried to product PHB in BL21(DE3)<br />
====PHB production test====<br />
We prepared those three medium tubes and incubated them for 48 hrs.<br />
#700 ml LB + 0.7 ul Amp<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose + 1.4 ul Sodium pantothenate<br />
*BL21(DE3) = No. 1~3 incubated<br />
*JM109 = No. 2 only incubated (control)<br />
<br />
Today the requested parts to HQ were sent! Parts were sent as agar stabs. We note here how to use these agar stabs.<br />
Using agar stab(http://partsregistry.org/Help:Requesting_Parts)<br />
We send out our part requests as agar stabs. The shelf life of these are short, so it is usually best to plate from the stab as soon as possible.<br /><br />
The agar should already have a hole from when it was stabbed. With an inoculating loop dipped into the stab, you can plate directly onto a petri dish with the appropriate antibiotic.<br />
#Incubate the dish overnight at 37C (14-16hrs)<br />
#Pick a single colony to start up a culture<br />
#Extract plasmid DNA<br />
#Use the part! <br />
<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_2
Team:HokkaidoU Japan/Notebook/plastic Week 2
2012-09-27T03:55:39Z
<p>Kwt: /* Transformation */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===July 9th===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
Transformation of pGEM into JM109. This transformation is the '''bioplastic production test in JM109'''.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto LBA dish and spread.<br />
#Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBA dish then spread. <br />
#Incubated the plates at 37C for 15 hrs 30 min.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===July 10th===<br />
<div class="hokkaidou-section"><br />
====Incubation====<br />
#Single colony isolation.<br />
#Pre-culture with 2xLB/A for 10~14 hrs, 180 rpm, 30C .<br />
#Cultured in plastic producing media for 48hrs, 180 rpm.<br />
<br />
====Polymer extract====<br />
Measure the weight of tube.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===July 14th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Extracted the plasmid.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_2
Team:HokkaidoU Japan/Notebook/plastic Week 2
2012-09-27T03:54:48Z
<p>Kwt: /* Incubation */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===July 9th===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
Transformation of pGEM into JM109. This transformation is the '''bioplastic production test in JM109'''.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto LBA dish and spread.<br />
#Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBA dish then spread. <br />
#Incubated the plates at 37C for 15hrs 30 min.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===July 10th===<br />
<div class="hokkaidou-section"><br />
====Incubation====<br />
#Single colony isolation.<br />
#Pre-culture with 2xLB/A for 10~14 hrs, 180 rpm, 30C .<br />
#Cultured in plastic producing media for 48hrs, 180 rpm.<br />
<br />
====Polymer extract====<br />
Measure the weight of tube.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===July 14th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Extracted the plasmid.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_5
Team:HokkaidoU Japan/Notebook/plastic Week 5
2012-09-27T03:53:21Z
<p>Kwt: /* Transformation */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===August 2nd===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
We transformed pGEM into E.coli (strain:BL21). This transformation is the '''bioplastic production test in BL21'''.<br />
#Added 1 ul of plasmid DNA solution to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto LBA dish and spread.<br />
#Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBA dish then spread. <br />
#Incubated the plates at 37C for 15 hrs 30 min.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===August 3rd===<br />
<div class="hokkaidou-section"><br />
====Liquid culture====<br />
Liquid culture of transformed BL21(DE3)<br />
#Added 2 ml of LBA into culture tubes.<br />
#Resuspended 2 colonies.<br />
#Incubated the tubes at 37C for OOhrs.<br />
<br />
After incubating, we tried to product PHB in BL21(DE3)<br />
====PHB production test====<br />
We prepared those three medium tubes and incubated them for OOhrs.<br />
#700 ml LB + 0.7 ul Amp<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose<br />
#700 ml LB + 0.7 ul Amp + 28 ul 50% glucose + 1.4 ul Sodium pantothenate<br />
*BL21(DE3) = No. 1~3 incubated<br />
*JM109 = No. 2 only incubated (control)<br />
<br />
Today the requested parts to HQ were sent! Parts were sent as agar stabs. We note here how to use these agar stabs.<br />
Using agar stab(http://partsregistry.org/Help:Requesting_Parts)<br />
We send out our part requests as agar stabs. The shelf life of these are short, so it is usually best to plate from the stab as soon as possible.<br /><br />
The agar should already have a hole from when it was stabbed. With an inoculating loop dipped into the stab, you can plate directly onto a petri dish with the appropriate antibiotic.<br />
#Incubate the dish overnight at 37C (14-16hrs)<br />
#Pick a single colony to start up a culture<br />
#Extract plasmid DNA<br />
#Use the part! <br />
<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:21:28Z
<p>Kwt: /* HPLC test */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
===Staining by Nile red===<br />
<p><br />
<br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
Specific signal associated with interaction between Nile red an P3HB was detected only in the presence of plastic producing module(right panel in figure5), but not in the absence of it(left panel in figure 4)<br />
<br />
[[image:HokkaidoU2012_Nilered.jpg|center|thumb|600px|hogehoge]]<br />
<br />
</p><br />
<br />
===HPLC test===<br />
<br />
<p><br />
We confirmed the existence of P3HB by analyzing building block of it after hydrolysis of P3HB by using HPLC.<br />
Degraded building block of P3HB was detected in E. coli containing both "plastic producing module" and "Aggregation module".<br />
<br />
<br />
Result indicates that in<br />
<br />
<br />
A=added only arabinose G=added only glucose AG=addded both arabinose and glucose.<br />
<br />
<br />
<br />
<br />
[[image:pGEM+Ag (A G AG).jpg|center|thumb|350px|Result of HPLC test]]<br />
<br />
<br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:16:05Z
<p>Kwt: /* HPLC test */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
===Staining by Nile red===<br />
<p><br />
<br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
Specific signal associated with interaction between Nile red an P3HB was detected only in the presence of plastic producing module(right panel in figure5), but not in the absence of it(left panel in figure 4)<br />
<br />
[[image:HokkaidoU2012_Nilered.jpg|center|thumb|600px|hogehoge]]<br />
<br />
</p><br />
<br />
===HPLC test===<br />
<br />
<p><br />
We confirmed <br />
<br />
<br />
<br />
[[image:pGEM+Ag (A G AG).jpg|center|thumb|350px|Result of HPLC test]]<br />
<br />
<br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:13:15Z
<p>Kwt: /* HPLC test */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
===Staining by Nile red===<br />
<p><br />
<br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
Specific signal associated with interaction between Nile red an P3HB was detected only in the presence of plastic producing module(right panel in figure5), but not in the absence of it(left panel in figure 4)<br />
<br />
[[image:HokkaidoU2012_Nilered.jpg|center|thumb|600px|hogehoge]]<br />
<br />
</p><br />
<br />
===HPLC test===<br />
<br />
<p><br />
We confirmed <br />
<br />
<br />
<br />
[[image:pGEM+Ag (A G AG).jpg|center|thumb|350px|HPLC result]]<br />
<br />
<br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/File:PGEM%2BAg_(A_G_AG).jpg
File:PGEM+Ag (A G AG).jpg
2012-09-27T03:12:36Z
<p>Kwt: </p>
<hr />
<div></div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:12:18Z
<p>Kwt: /* HPLC test */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
===Staining by Nile red===<br />
<p><br />
<br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
Specific signal associated with interaction between Nile red an P3HB was detected only in the presence of plastic producing module(right panel in figure5), but not in the absence of it(left panel in figure 4)<br />
<br />
[[image:HokkaidoU2012_Nilered.jpg|center|thumb|600px|hogehoge]]<br />
<br />
</p><br />
<br />
===HPLC test===<br />
<br />
<p><br />
We confirmed <br />
<br />
<br />
<br />
[[image:pGEM+Ag (A G AG).jpg|center|thumb|700px|HPLC result]]<br />
<br />
<br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T03:11:34Z
<p>Kwt: /* HPLC test */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
===Staining by Nile red===<br />
<p><br />
<br />
We identified the production of plastic by conventional staining method (staining with Nile red) in E. coli containing both "plastic producing module" and "Aggregation module".<br />
Specific signal associated with interaction between Nile red an P3HB was detected only in the presence of plastic producing module(right panel in figure5), but not in the absence of it(left panel in figure 4)<br />
<br />
[[image:HokkaidoU2012_Nilered.jpg|center|thumb|600px|hogehoge]]<br />
<br />
</p><br />
<br />
===HPLC test===<br />
<br />
<p><br />
We confirmed <br />
<br />
<br />
<br />
[[image:pGEM+Ag (A G AG).png|center|thumb|700px|HPLC result]]<br />
<br />
<br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/File:HokkaidoU_IMG_2463.JPG
File:HokkaidoU IMG 2463.JPG
2012-09-27T02:55:51Z
<p>Kwt: uploaded a new version of &quot;File:HokkaidoU IMG 2463.JPG&quot;</p>
<hr />
<div></div>
Kwt
http://2012.igem.org/File:HokkaidoU_IMG_2463.JPG
File:HokkaidoU IMG 2463.JPG
2012-09-27T02:53:43Z
<p>Kwt: uploaded a new version of &quot;File:HokkaidoU IMG 2463.JPG&quot;</p>
<hr />
<div></div>
Kwt
http://2012.igem.org/File:HokkaidoU_IMG_2463.JPG
File:HokkaidoU IMG 2463.JPG
2012-09-27T02:47:38Z
<p>Kwt: uploaded a new version of &quot;File:HokkaidoU IMG 2463.JPG&quot;</p>
<hr />
<div></div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project/Biocapsule
Team:HokkaidoU Japan/Project/Biocapsule
2012-09-27T02:44:44Z
<p>Kwt: /* Results */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<br />
==Introduction==<br />
<br />
<p><br />
The aim of our project was to make a smart system for industrial production of high-value added macromolecules by using BioDevice function in E. coli. We call it, “Bio capsule” method. At the beginning, we expected to be able to make “Bio-capsule” only by employing a function of “Aggregation module”, however, resultant E. coli cluster was not sufficiently hard enough to be recovered on filter through nylon mesh filtration. However we accidentally found that co-expression of both “Plastic Producing Module” and “Aggregation Module” results in forming hard and heavy E. coli clusters which can be called real “Bio capsule” we expected.<br />
</p><br />
<br />
==Method==<br />
<br />
<p><br />
We used two kinds of compatible plasmid vector to make E. coli expresses both Ag43 and all enzymes required for P3HB production. We chose pSB1C3 plasmid vector, a high copy number plasmid vector containing replication origin from R- factor, for the expression of “Plastic Producing Module” to produce enough amount of bio-plastic. We chose pSTV28 plasmid vector that contains compatible replication origin to pSB series: the most popular plasmid vector in iGEM, for expression “Aggregation Module”. It is widely known that if there were two similar plasmids those containing same replication origin in single cell, they compete with each other for replication and only one of which can be amplified in E. coli. <br /><br />
<br />
<br />
[[image:HokkaidoU Bio capsule module.2.png|center|thumb|700px|Fig1 Image of our Bio capsule]]<br />
<br />
<br />
As a pilot experiment, we made E. coli containing plasmid for “Aggregation module” and plasmid for “Plastic producing module”. “Aggregation module” is induced by L-arabinose, since Ag43 is expressed under control of PBAD promoter. “Plastic producing module” used in the experiment in table 1 and 2 is expressed under control of original promoter of gene cluster encoding all enzymes required for P3HB production in R. eutropha.<br /><br />
<br />
<br />
</p><br />
<br />
==Results==<br />
<p><br />
The aggregation is induced by the addition of arabinose for 1%, and production of P3HB is initiated by the addition of glucose for 2% as energy source, so adding both chemicals to the culture media initiates thttps://2012.igem.org/wiki/skins/common/images/button_italic.pngo start expression of both modules. Hard and heavy E. coli clusters were observed only in a presence of these chemicals as shown in Table1.<br />
<br />
<br />
[[image:HokkaidoU Table1.jpg|center|thumb|800px|Table1]]<br />
<br />
[[image:HokkaidoU2012_24h-48h.jpg|center|thumb|800px|figure2]]<br />
<br />
<br />
<br />
Figure2 shows photographic image of the culture tested. Each condition is indicated below each image. After cultivation for 24hours, each three culture media did not have big difference (panel A in figure 2). However, after cultivation for 48hours, we could clearly observe that in the second sample (2%glucose (+) 1%Arabinose (+)), its supernatant had high clarity than others (panel B in figure 2). <br /><br />
Furthermore, hard and heavy clusters of cells were sticking to the surface of side wall in the test tube and precipitating in the media (fig3).<br />
<br />
[[image:HokkaidoU IMG 2463.JPG|center|thumb|1000px|Figure3 clustered cells]]<br />
<br />
<br />
</p><br />
<br />
===precipitation test===<br />
[[image:Hokkaido Uver20min &amp; 5min.jpg|center|thumb|500px|0min & 5min]]<br />
<br />
[[image:HokkaidoU10min &amp;15min.jpg|center|thumb|500px|10min & 15min]]<br />
<br />
<br />
[[image:HokkaidoU30min &amp; 1hr.jpg|center|thumb|500px|30min & 1hrs]]<br />
<p><br />
The speed of precipitation was measured.<br />
The photograph of media was taken at the timing of 0min, 5min, 10min, 15min, 30min, and 1hr.<br />
<br />
As you can see, the media with the expression of both Ag43 and phaCAB, had the fastest precipitation.<br />
<br />
</p><br />
<br />
==Discussion(Improvement)==<br />
<p><br />
Our finding of method to create “Hard and heavy cluster” when both Ag43 and phaCAB was expressed, was an unexpected result. However, this result we got was a positive one, and we can say it is our team’s serendipity. <br />
When we expressed Ag43 only, the cell makes a weak cluster which is not suitable for harvesting “Plastic producing module”. However, when we combine two modules intoa single cell, and induce both of their expressions, the cells produces “Hard and heavy cluster” which fits for our image of “Bio-capsule”. The expression of plastics makes the cells heavier than wild type E. coli and results to precipitate the cells faster than ever. In addition, the plastic have acted as a glue to reinforce the attachment of Ag43 protein locating on the surface of E. coli cells.<br />
</p><br />
<br />
==Future planning==<br />
<p> <br />
Our result indicates the success of collecting cells by filtration or by decantation in large scales like manufacturing. When manufacturing bio plastics by bacteria, the method of harvesting is indispensable. However, the method of centrifugation is time and cost consuming, especially when the scale gets larger. In contrast, with the usage with our bio capsule module, the method of harvesting turns into an easy and low costing job. Our success may contribute to future manufacturing’s bio plastics by bacteria.<br />
In addition, the candidate inside of the bio capsule is not limited to P3HB. We have various kinds of plastics that are useful. For example, P4HB(4-Hydroxybutyrate) described as a strong pliable thermoplastic material is used for surgery strings because it is elongate and bio degradable. Furthermore, P4HB could form co-polymers with P3HB. If we succeed to submit the enzymes relating to the synthesis of P4HB, we will be able to construct an bio capsule which contains P4HB.<br />
</p><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/File:HokkaidoU_IMG_2463.JPG
File:HokkaidoU IMG 2463.JPG
2012-09-27T02:35:49Z
<p>Kwt: uploaded a new version of &quot;File:HokkaidoU IMG 2463.JPG&quot;</p>
<hr />
<div></div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook
Team:HokkaidoU Japan/Notebook
2012-09-26T23:56:26Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
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height:100px;<br />
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-webkit-transition:all 0.2s ease;<br />
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line-height:1.5;<br />
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background-position: 673px -781px;<br />
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background-position: 543px -781px;<br />
}<br />
<br />
</style><br />
<h2>Notebook</h2><br />
<div class="hokkaidou-section"><br />
<a href="/Team:HokkaidoU_Japan/Notebook/Spring_Boot_Camp" class="hokkaidou-notebook-index" id="hokkaidou-notebook-bootcamp"><br />
<h5>Boot Camp</h5><br />
<p>Be my apprentice.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Notebook/Lab_Diary" class="hokkaidou-notebook-index" id="hokkaidou-notebook-diary"><br />
<h5>Lab Diary</h5><br />
<p>That's one small step for you, one giant leap for us. </p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Notebook/overall_protocols" class="hokkaidou-notebook-index" id="hokkaidou-notebook-protocols"><br />
<h5>Protocols</h5><br />
<p>Bible of our lab.</p><br />
</a><br />
</div><br />
</html><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook
Team:HokkaidoU Japan/Notebook
2012-09-26T23:47:53Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
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.hokkaidou-notebook-index {<br />
margin:0 0 10px 0;<br />
display:block;<br />
height:100px;<br />
padding:15px;<br />
background: rgba(255, 255, 255, 0.2);<br />
color:#b2aaa4;<br />
-webkit-transition:all 0.2s ease;<br />
-moz-transition:all 0.2s ease;<br />
background-repeat:no-repeat;<br />
text-decoration: none !important;<br />
border-radius: 1em;<br />
}<br />
.hokkaidou-notebook-index h5 {<br />
font-size:24px;<br />
line-height:1;<br />
padding:0 0 10px 0;<br />
}<br />
.hokkaidou-notebook-index h5 {<br />
color:white;<br />
}<br />
.hokkaidou-notebook-index:hover p {<br />
color:white;<br />
}<br />
.hokkaidou-notebook-index p {<br />
font-size:12px;<br />
width:500px;<br />
line-height:1.5;<br />
color: #ffffff;<br />
}<br />
.hokkaidou-notebook-index:hover {<br />
background-position:200px 50%;<br />
}<br />
#hokkaidou-notebook-bootcamp {<br />
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background-position: 673px -521px;<br />
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background-position: 543px -521px;<br />
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#hokkaidou-notebook-diary {<br />
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background-position: 673px -651px;<br />
}<br />
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background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');<br />
background-position: 673px -781px;<br />
}<br />
#hokkaidou-notebook-protocols:hover {<br />
background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');<br />
background-position: 543px -781px;<br />
}<br />
<br />
</style><br />
<h2>Notebook</h2><br />
<div class="hokkaidou-section"><br />
<a href="/Team:HokkaidoU_Japan/Notebook/Spring_Boot_Camp" class="hokkaidou-notebook-index" id="hokkaidou-notebook-bootcamp"><br />
<h5>Boot Camp</h5><br />
<p>Be my apprentice.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Notebook/Lab_Diary" class="hokkaidou-notebook-index" id="hokkaidou-notebook-diary"><br />
<h5>Lab Diary</h5><br />
<p>Our lots of work in everyday must be a little step to championship!! </p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Notebook/overall_protocols" class="hokkaidou-notebook-index" id="hokkaidou-notebook-protocols"><br />
<h5>Protocols</h5><br />
<p>Bible of our lab.</p><br />
</a><br />
</div><br />
</html><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project
Team:HokkaidoU Japan/Project
2012-09-26T23:29:44Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.project}}<br />
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<style type="text/css"><br />
.hokkaidou-project-index {<br />
margin:0 0 10px 0;<br />
display:block;<br />
height:100px;<br />
padding:15px;<br />
background: rgba(255, 255, 255, 0.2);<br />
color:#b2aaa4;<br />
-webkit-transition:all 0.2s ease;<br />
-moz-transition:all 0.2s ease;<br />
background-repeat:no-repeat;<br />
text-decoration: none !important;<br />
border-radius: 1em;<br />
}<br />
.hokkaidou-project-index h5 {<br />
font-size:24px;<br />
line-height:1;<br />
padding:0 0 10px 0;<br />
}<br />
.hokkaidou-project-index h5 {<br />
color:white;<br />
}<br />
.hokkaidou-project-index:hover p {<br />
color:white;<br />
}<br />
.hokkaidou-project-index p {<br />
font-size:12px;<br />
width: 500px;<br />
line-height:1.5;<br />
color: #ffffff;<br />
}<br />
.hokkaidou-project-index:hover {<br />
background-position:200px 50%;<br />
}<br />
<br />
#hokkaidou-project-aggregation {<br />
background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');<br />
background-position: 673px 0;<br />
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background-position: 673px -130px;<br />
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background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');<br />
background-position: 673px -390px;<br />
}<br />
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background-image: url('https://static.igem.org/mediawiki/2012/e/ea/HokkaidoU2012_Menu_Circle.png');<br />
background-position: 543px -390px;<br />
}<br />
<br />
</style><br />
<h2>Project</h2><br />
<div class="hokkaidou-section"><br />
<a href="/Team:HokkaidoU_Japan/Project/Aggregation" class="hokkaidou-project-index" id="hokkaidou-project-aggregation"><br />
<h5>Aggregation Module</h5><br />
<p>You have no need to centrifuge anymore.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/PHB_Synthesis" class="hokkaidou-project-index" id="hokkaidou-project-phb"><br />
<h5>PHB Synthesis</h5><br />
<p>PHB has possibility to be utilized in various fields.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/Biocapsule" class="hokkaidou-project-index" id="hokkaidou-project-capsule"><br />
<h5>Biocapsule</h5><br />
<p>What happens when these two modules work?</p><br />
</a><br />
</div><br />
</html><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="clear: both; line-height: 0;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:27:44Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
<br />
====Liquid culture====<br />
We got colonies on the plate, and started liquid culture.<br />
<br />
====PCR====<br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
<br />
====Digestion====<br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
<br />
====Gel extraction====<br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ethanol precipitation====<br />
The digested DNAs were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
<br />
====Transformation====<br />
Transformed each ligation product into JM109, and incubated.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
Colony PCR for colonies transformed at 25th.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:13:27Z
<p>Kwt: /* Single Colony isolation */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
<br />
====Liquid culture====<br />
We got colonies on the plate, and started liquid culture.<br />
<br />
====PCR====<br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
<br />
====Digestion====<br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
<br />
====Gel extraction====<br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ligation====<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
====Transformation====<br />
Transformed each ligation product into JM109, and incubated.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
Colony PCR for colonies transformed at 25th.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:13:10Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
<br />
====Liquid culture====<br />
We got colonies on the plate, and started liquid culture.<br />
<br />
====PCR====<br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
<br />
====Digestion====<br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
<br />
====Gel extraction====<br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ligation====<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
====Transformation====<br />
Transformed each ligation product into JM109, and incubated.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
Colony PCR for colonies transformed at 25th.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_12
Team:HokkaidoU Japan/Notebook/plastic Week 12
2012-09-26T22:11:57Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 17th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Plasmids of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 and pTet(BBa_0040) on pSB1A2 were extracted.<br/><br />
And then we got DNA solution of them.<br />
<br />
====Digestion====<br />
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested by XbaI and PstI.<br/><br />
pTet on pSB1A2 was also digested by SpeI and PstI.<br />
<br />
====Gel extraction====<br />
We confirmed the succession of digestion by electrophoresis.<br/><br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ethanol precipitation====<br />
The digested DNAs were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was ligated with pTet on pSB1A2.<br />
<br />
====Transformation====<br />
The ligated DNA was transformed into E. coli (strain: JM109).<br/><br />
And then we spread fungus liquid on LBA plates.<br />
<br />
====Colony PCR====<br />
We confirmed the ligation of [RBS-B] and pSB1C3.<br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
<br />
====Liquid culture====<br />
The colonies of [RBS-phaC-RBS-phaA on pSB1A2] and [RBS-phaC-RBS-phaA-RBS-phaC-dT on pSB1A2] were condensed in LB.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 18th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
We confirmed the succession of ligation RBS-PhaC-RBS-PhaA-RBS-PhaB-dT with pTet on pSB1A2 by colony PCR.<br/><br />
RBS-PhaB-dT, a part of insert was multiplied.<br />
<br />
====Sequencing====<br />
The sequence of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was analyzed.<br />
The result showed that...<br />
<br />
====Plasmid extraction====<br />
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2], [RBS-PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] were extracted.<br />
<br />
====Digestion====<br />
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and XbaI.<br><br />
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested by EcoRI and PstI.<br><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br><br />
But we failed the second digestion. <br />
<br />
====Gel extraction====<br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ethanol precipitation====<br />
The extracted DNAs were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated.<br />
<br />
====Liquid culture====<br />
We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180 rpm to digest and ligate again.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 19th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] were extracted.<br />
<br />
====Digestion====<br />
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and SpeI.<br><br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested by EcoRI and PstI.<br><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br><br />
<br />
====DNA extraction====<br />
The digested DNA were extracted.<br />
<br />
====Ethanol precipitation====<br />
The extracted DNA were condensed by EtOH precipitation.<br />
<br />
====Ligation====<br />
[RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3] were ligated.<br><br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated too.<br />
<br />
====Transformation====<br />
The ligated DNA were transformed into E. coli (strain: JM109).<br/><br />
E. coli solution was spread on LBC.<br />
<br />
====Colony PCR====<br />
We confirmed the ligation [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/><br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 20th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
We confirmed the ligation [RBS-PhaC-RBS-PhaA] with [RBS-PhaB on pSB1C3] and<br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/><br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 21st===<br />
<div class="hokkaidou-section"><br />
<br />
===Plasmid extraction===<br />
Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted.<br />
<br />
We submitted our parts and finished sending them !<br />
Hooray!;)<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 22nd===<br />
<div class="hokkaidou-section"><br />
====Liquid culture in large scale====<br />
We made cells which contains both plasmids of phaCAB on pGEM and Ag43 on pSTV28.<br />
Ab43 are induced by arabinose, and the production of P3HB is induced by glucose.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|LB<br />
|mess up to 50 ml<br />
|-<br />
|50% Glucose<br />
|2 ml<br />
|-<br />
|Arabinose<br />
|2.5 ml<br />
|-<br />
|Amp(100 mg/ml)<br />
|50 ul<br />
|-<br />
|Cp<br />
|50 ul<br />
|-<br />
|}<br />
<br />
Negative control<br />
#Gulcose (-)<br />
#Arabinose (-)<br />
<br />
Cultured for 48 hrs.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 23rd===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
Transformed BBa_R0011 for ligation of promoter with IPTG induction.<br />
<br />
Incubated in LB+K plate.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_12
Team:HokkaidoU Japan/Notebook/plastic Week 12
2012-09-26T22:11:04Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 17th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Plasmids of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 and pTet(BBa_0040) on pSB1A2 were extracted.<br/><br />
And then we got DNA solution of them.<br />
<br />
====Digestion====<br />
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested by XbaI and PstI.<br/><br />
pTet on pSB1A2 was also digested by SpeI and PstI.<br />
<br />
====Gel extraction====<br />
We confirmed the succession of digestion by electrophoresis.<br/><br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ethanol precipitation====<br />
The digested DNAs were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was ligated with pTet on pSB1A2.<br />
<br />
====Transformation====<br />
The ligated DNA was transformed into E. coli (strain: JM109).<br/><br />
And then we spread fungus liquid on LBA plates.<br />
<br />
====Colony PCR====<br />
We confirmed the ligation of [RBS-B] and pSB1C3.<br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
<br />
====Liquid culture====<br />
The colonies of [RBS-phaC-RBS-phaA on pSB1A2] and [RBS-phaC-RBS-phaA-RBS-phaC-dT on pSB1A2] were condensed in LB.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 18th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
We confirmed the succession of ligation RBS-PhaC-RBS-PhaA-RBS-PhaB-dT with pTet on pSB1A2 by colony PCR.<br/><br />
RBS-PhaB-dT, a part of insert was multiplied.<br />
<br />
====Sequencing====<br />
The sequence of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was analyzed.<br />
The result showed that...<br />
<br />
====Plasmid extraction====<br />
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2], [RBS-PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] were extracted.<br />
<br />
====Digestion====<br />
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and XbaI.<br><br />
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested by EcoRI and PstI.<br><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br><br />
But we failed the second digestion. <br />
<br />
====Gel extraction====<br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ethanol precipitation====<br />
The extracted DNAs were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated.<br />
<br />
====Liquid culture====<br />
We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180 rpm to digest and ligate again.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 19th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] were extracted.<br />
<br />
====Digestion====<br />
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and SpeI.<br><br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested by EcoRI and PstI.<br><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br><br />
<br />
====DNA extraction====<br />
The digested DNA were extracted.<br />
<br />
====Ethanol precipitation====<br />
The extracted DNA were condensed by EtOH precipitation.<br />
<br />
====Ligation====<br />
[RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3] were ligated.<br><br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated too.<br />
<br />
====Transformation====<br />
The ligated DNA were transformed into E. coli (strain: JM109).<br/><br />
E. coli solution was spread on LBC.<br />
<br />
====Colony PCR====<br />
We confirmed the ligation [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/><br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 20th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
We confirmed the ligation [RBS-PhaC-RBS-PhaA] with [RBS-PhaB on pSB1C3] and<br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/><br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 21st===<br />
<div class="hokkaidou-section"><br />
<br />
===Plasmid extraction===<br />
Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted.<br />
<br />
We submitted our parts and finished sending them !<br />
Hooray!;)<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 22th===<br />
<div class="hokkaidou-section"><br />
====Liquid culture in large scale====<br />
We made cells which contains both plasmids of phaCAB on pGEM and Ag43 on pSTV28.<br />
Ab43 are induced by arabinose, and the production of P3HB is induced by glucose.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|LB<br />
|mess up to 50 ml<br />
|-<br />
|50% Glucose<br />
|2 ml<br />
|-<br />
|Arabinose<br />
|2.5 ml<br />
|-<br />
|Amp(100 mg/ml)<br />
|50 ul<br />
|-<br />
|Cp<br />
|50 ul<br />
|-<br />
|}<br />
<br />
Negative control<br />
#Gulcose (-)<br />
#Arabinose (-)<br />
<br />
Cultured for 48 hrs.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 23th===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
Transformed BBa_R0011 for ligation of promoter with IPTG induction.<br />
<br />
Incubated in LB+K plate.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:09:31Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
<br />
====Liquid culture====<br />
We got colonies on the plate, and started liquid culture.<br />
<br />
====PCR====<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
====Digestion====<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
====Gel extraction====<br />
<p><br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
</p><br />
<br />
====Ligation====<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
====Transformation====<br />
<p><br />
Transformed each ligation product into JM109, and incubated.<br />
</p><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:07:50Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
====PCR====<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
====Digestion====<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
====Gel extraction====<br />
<p><br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
</p><br />
<br />
====Ligation====<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
====Transformation====<br />
<p><br />
Transformed each ligation product into JM109, and incubated.<br />
</p><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:05:48Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
====PCR====<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
====Digestion====<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
====Ligation====<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
====Transformation====<br />
<p><br />
Transformed each ligation product into JM109, and incubated.<br />
</p><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:04:57Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
==PCR==<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
==Digestion==<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformed each ligation product into JM109, and incubated.<br />
</p><br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:04:17Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
==PCR==<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
==Digestion==<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformed each ligation product into JMi09, and incubated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 26th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:03:20Z
<p>Kwt: /* September 26th */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
==PCR==<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
==Digestion==<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformed each ligation product into JMi09, and incubated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 26th===<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:03:03Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
==PCR==<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
==Digestion==<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformed each ligation product into JMi09, and incubated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==September 26th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T22:02:40Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
==PCR==<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
==Digestion==<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformed each ligation product into JMi09, and incubated.<br />
</p><br />
</div></div><br />
#Incuvated.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==September 26th==<br />
<div><br />
===Colony PCR===<br />
<p><br />
Colony PCR for colonies transformed at 25th.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_13
Team:HokkaidoU Japan/Notebook/plastic Week 13
2012-09-26T21:58:33Z
<p>Kwt: /* September 25th */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 24th===<br />
<div class="hokkaidou-section"><br />
<br />
====Single Colony isolation====<br />
We got few colonies from the plate.<br />
The colonies were isolated to another plate.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 25th===<br />
<div class="hokkaidou-section"><br />
We got colonies on the plate, and started liquid culture.<br />
<br />
==PCR==<br />
<p><br />
Did PCR for promoter of R.eutropha from pGEM to ligate with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2.<br />
</p> <br />
<br />
==Digestion==<br />
<p><br />
Digested the promoter done PCR and RBS-PhaA on pSB1A2 with XbaI and SpeI, and plasmid of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 with XbaI.<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligated the promoter with RBS-PhaC-RBS-PhaA-RBS-PhaB-dT and with pSB1A2.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformed each ligation product into JMi09, and incubated.<br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/plastic_Week_12
Team:HokkaidoU Japan/Notebook/plastic Week 12
2012-09-26T21:46:13Z
<p>Kwt: /* Transformation */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
===September 17th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Plasmids of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 and pTet(BBa_0040) on pSB1A2 were extracted.<br/><br />
And then we got DNA solution of them.<br />
<br />
====Digestion====<br />
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2 was digested by XbaI and PstI.<br/><br />
pTet on pSB1A2 was also digested by SpeI and PstI.<br />
<br />
====Gel extraction====<br />
We confirmed the succession of digestion by electrophoresis.<br/><br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ethanol precipitation====<br />
The digested DNAs were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was ligated with pTet on pSB1A2.<br />
<br />
====Transformation====<br />
The ligated DNA was transformed into E. coli (strain: JM109).<br/><br />
And then we spread fungus liquid on LBA plates.<br />
<br />
====Colony PCR====<br />
We confirmed the ligation of [RBS-B] and pSB1C3.<br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
<br />
====Liquid culture====<br />
The colonies of [RBS-phaC-RBS-phaA on pSB1A2] and [RBS-phaC-RBS-phaA-RBS-phaC-dT on pSB1A2] were condensed in LB.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 18th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
We confirmed the succession of ligation RBS-PhaC-RBS-PhaA-RBS-PhaB-dT with pTet on pSB1A2 by colony PCR.<br/><br />
RBS-PhaB-dT, a part of insert was multiplied.<br />
<br />
====Sequencing====<br />
The sequence of RBS-PhaC-RBS-PhaA-RBS-PhaB-dT was analyzed.<br />
The result showed that...<br />
<br />
====Plasmid extraction====<br />
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2], [RBS-PhaB on pSB1C3] and [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] were extracted.<br />
<br />
====Digestion====<br />
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and XbaI.<br><br />
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT on pSB1A2] was digested by EcoRI and PstI.<br><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br><br />
But we failed the second digestion. <br />
<br />
====Gel extraction====<br />
We confirmed succession of digestion by electrophoresis.<br />
And then DNA were extracted from TBE gel.<br />
<br />
====Ethanol precipitation====<br />
The extracted DNAs were condensed by Ethanol precipitation.<br />
<br />
====Ligation====<br />
[RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated.<br />
<br />
====Liquid culture====<br />
We started to incubate the colony of [RBS-PhaC-RBS-PhaA on pSB1A2] at 37C, 180 rpm to digest and ligate again.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 19th===<br />
<div class="hokkaidou-section"><br />
====Plasmid extraction====<br />
Plasmids of [RBS-PhaC-RBS-PhaA on pSB1A2] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] were extracted.<br />
<br />
====Digestion====<br />
[RBS-PhaC-RBS-PhaA on pSB1A2] was digested by EcoRI and SpeI restriction sites.<br/><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and SpeI.<br><br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1A2] was digested by EcoRI and PstI.<br><br />
[RBS-PhaB on pSB1C3] was digested by EcoRI and PstI.<br><br />
<br />
====DNA extraction====<br />
The digested DNA were extracted.<br />
<br />
====Ethanol precipitation====<br />
The extracted DNA were condensed by EtOH precipitation.<br />
<br />
====Ligation====<br />
[RBS-PhaC-RBS-PhaA] and [RBS-PhaB on pSB1C3] were ligated.<br><br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] and pSB1C3 were ligated too.<br />
<br />
====Transformation====<br />
The ligated DNA were transformed into E. coli (strain: JM109).<br/><br />
E. coli solution was spread on LBC.<br />
<br />
====Colony PCR====<br />
We confirmed the ligation [RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/><br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 20th===<br />
<div class="hokkaidou-section"><br />
====Colony PCR====<br />
We confirmed the ligation [RBS-PhaC-RBS-PhaA] with [RBS-PhaB on pSB1C3] and<br />
[pTet-RBS-PhaC-RBS-PhaA-RBS-PhaB-dT] with [pSB1C3] by colony PCR.<br/><br />
The results showed that the ligation went well.<br />
<br />
We chose three colonies and started the liquid culture.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
===September 21st===<br />
<div class="hokkaidou-section"><br />
<br />
===Plasmid extraction===<br />
Plasmids of [RBS-PhaC-RBS-PhaA-RBS-PhaB on pSB1C3] and [pTet-RBS-PhaC-RBS-PhaA-RBS-PhaC-dT on pSB1C3] were extracted.<br />
<br />
We submitted our parts and finished sending them !<br />
Hooray!;)<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
===September 22th===<br />
<div class="hokkaidou-section"><br />
====Liquid culture in large scale====<br />
We made cells which contains both plasmids of phaCAB on pGEM and Ag43 on pSTV28.<br />
Ab43 are induced by arabinose, and the production of P3HB is induced by glucose.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|LB<br />
|mess up to 50 ml<br />
|-<br />
|50% Glucose<br />
|2 ml<br />
|-<br />
|Arabinose<br />
|2.5 ml<br />
|-<br />
|Amp(100 mg/ml)<br />
|50 ul<br />
|-<br />
|Cp<br />
|50 ul<br />
|-<br />
|}<br />
<br />
Negative control<br />
#Gulcose (-)<br />
#Arabinose (-)<br />
<br />
Cultured for 48 hrs.<br />
</div></div><br />
<br />
===September 23th===<br />
<div class="hokkaidou-section"><br />
====Transformation====<br />
Transformed BBa_R0011 for ligation of promoter with IPTG induction.<br />
<br />
Incubated in LB+K plate.<br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_12
Team:HokkaidoU Japan/Notebook/aggregation Week 12
2012-09-26T21:43:20Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==September 17th==<br />
<div><br />
==Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2==<br />
<p><br />
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI.<br />
<br /><bbr /><br />
Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (45~50 ng/ul)<br />
|27 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|40 ul<br />
|}<br />
<br />
<br />
Vector(ptet-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
We confirmed that the concentration of Insert DNA solution is 60~70 ng/ul and Vector DNA solution is about 20~30 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (60~70 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (20~30 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 17 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 18th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==<br />
<p><br />
<br />
We did colony PCR two times.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(200bp down primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-f4 primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 702bp.<br />
<br />
[[image:HokkaidoU2012 120918 colop pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120918 colop2 pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result 2]]<br />
<br />
<br />
Target products didn't exist in all samples. We noticed the reason why such results shown is that we used incorrect pSB1C3 DNA solution which isn't confirmed the sequence. We'll try the synthesis once more.<br />
</p><br />
<br />
==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI and pSB1C3 with EcoRI and SpeI.<br />
<br /><br /><br />
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 35ng/ul)<br />
|41 ul<br />
|-<br />
|EcoRI<br />
|2 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xH buffer<br />
|5 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
Vector(pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120916 Digestion Insert Vector Control.jpg |thumb|digestion result]]<br />
<br />
</p><br />
==Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120916 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA <br />
|4 ul<br />
|-<br />
|Vector DNA<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
Transformation ligation product into JM109.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 19th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
<br />
Colony PCR to confirm whether pSTV28 vettor has pBAS-RBs-eCFP-RBs-Ag43-dT as insert or not.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(200bp down primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-f4 primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 702bp.<br />
<br />
[[image:HokkaidoU2012 120919 colop2 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]<br />
<br />
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.<br />
</p><br />
<br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3 and pBAD-RBS-eCFP-RBS-Ag43-dI on pSB1C3==<br />
<p><br />
<br />
Colony PCR to confirm whether the constructs written above really have eCFP and Ag43 coding sites or not by using primers: one can anneal to a part of eCFP coding site and another can anneal to a part of Ag43 coding site.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(FP-F primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-R primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1, N2 (DW only) as controls.<br />
Desired product is about 452 bp and 697 bp respectively.<br />
<br />
[[image:HokkaidoU2012 120919 colop3 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]<br />
<br />
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 20th==<br />
<div><br />
<p><br />
==Digestion of ptet-RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 (DNA solution eluted from colony No.10, 12 respectively in ) and ptet-pSB1A2==<br />
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI. We prepared DNa solution derived from No. 10 and No. 12 colony selected selected by the result of colony PCR for 18th.<br />
<br /><br /><br />
Insert No.10 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Insert No.12 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(ptet-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120919 digestion InsertNo10-InsertNo12-Vector.jpg|thumb|digestion result]]<br />
<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120919 Ethapre InsertNo10-InsertNo12-Vector.jpg|thumb|ethanol precipitation result]]<br />
We decided to use No. 10 digestion product for ligation, and confirmed that the concentration of Insert DNA solution is 50 ng/ul and Vector DNA solution is about 20~30 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (50 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (20~30 ng/ul)<br />
|1.5 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 21th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(X-phaB-F primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaC-RBs-phaA-RBS-phaB-pSB1A2)as controls.<br />
Desired product is about 1500bp.<br />
<br />
[[image:HokkaidoU2012 120921 colop1 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120921 colop2 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result2]]<br />
<br />
<br />
Target products exist in almost all samples. We selected No. 1, 2 colony for incubation for plasmid extraction and No. 6, 12, 14 for storing at 4C. <br />
</p><br />
</div></div><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_12
Team:HokkaidoU Japan/Notebook/aggregation Week 12
2012-09-26T21:39:49Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==September 17th==<br />
<div><br />
==Digestion of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 and ptet-pSB1A2==<br />
<p><br />
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI.<br />
<br /><bbr /><br />
Insert (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (45~50 ng/ul)<br />
|27 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|40 ul<br />
|}<br />
<br />
<br />
Vector(ptet-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
We confirmed that the concentration of Insert DNA solution is 60~70 ng/ul and Vector DNA solution is about 20~30 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (60~70 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (20~30 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 17 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 18th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==<br />
<p><br />
<br />
We did colony PCR two times.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(200bp down primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-f4 primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 702bp.<br />
<br />
[[image:HokkaidoU2012 120918 colop pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120918 colop2 pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3.jpg|thumb|Colony PCR result 2]]<br />
<br />
<br />
Target products didn't exist in all samples. We noticed the reason why such results shown is that we used incorrect pSB1C3 DNA solution which isn't confirmed the sequence. We'll try the synthesis once more.<br />
</p><br />
<br />
==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pSB1C3==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI and pSB1C3 with EcoRI and SpeI.<br />
<br /><br /><br />
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 35ng/ul)<br />
|41 ul<br />
|-<br />
|EcoRI<br />
|2 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xH buffer<br />
|5 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
Vector(pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120916 Digestion Insert Vector Control.jpg |thumb|digestion result]]<br />
<br />
</p><br />
==Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120916 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA <br />
|4 ul<br />
|-<br />
|Vector DNA<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
Transformation ligation product into JM109.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 19th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
<br />
Colony PCR to confirm whether pSTV28 vettor has pBAS-RBs-eCFP-RBs-Ag43-dT as insert or not.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(200bp down primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-f4 primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 702bp.<br />
<br />
[[image:HokkaidoU2012 120919 colop2 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]<br />
<br />
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.<br />
</p><br />
<br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3 and pBAD-RBS-eCFP-RBS-Ag43-dI on pSB1C3==<br />
<p><br />
<br />
Colony PCR to confirm whether the constructs written above really have eCFP and Ag43 coding sites or not by using primers: one can anneal to a part of eCFP coding site and another can anneal to a part of Ag43 coding site.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(FP-F primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(ag43-R primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1, N2 (DW only) as controls.<br />
Desired product is about 452 bp and 697 bp respectively.<br />
<br />
[[image:HokkaidoU2012 120919 colop3 pBAD-RBS-eCFP-RBS-AG43-dT-pSTV28(pSB1C3).jpg|thumb|Colony PCR result]]<br />
<br />
We noticed the some of colones have pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 construct as plasmid DNA.<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 20th==<br />
<div><br />
<p><br />
==Digestion of ptet-RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 (DNA solution eluted from colony No.10, 12 respectively in ) and ptet-pSB1A2==<br />
To make a construct of ptet-RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 with XbaI and PstI, ptet-pSB1A2 with SpeI and PstI. We prepared DNa solution derived from No. 10 and No. 12 colony selected selected by the result of colony PCR for 18th.<br />
<br />
Insert No.10 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Insert No.12 (RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|4 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(ptet-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120919 digestion InsertNo10-InsertNo12-Vector.jpg|thumb|digestion result]]<br />
<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120919 Ethapre InsertNo10-InsertNo12-Vector.jpg|thumb|ethanol precipitation result]]<br />
We decided to use No. 10 digestion product for ligation, and confirmed that the concentration of Insert DNA solution is 50 ng/ul and Vector DNA solution is about 20~30 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (50 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (20~30 ng/ul)<br />
|1.5 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 21th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-Ag43-dI-pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(X-phaB-F primer)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaC-RBs-phaA-RBS-phaB-pSB1A2)as controls.<br />
Desired product is about 1500bp.<br />
<br />
[[image:HokkaidoU2012 120921 colop1 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result]]<br />
[[image:HokkaidoU2012 120921 colop2 ptet-RBS-phaCAB-RBS-eYFP-dT-pSB1C3.jpg|thumb|Colony PCR result2]]<br />
<br />
<br />
Target products exist in almost all samples. We selected No. 1, 2 colony for incubation for plasmid extraction and No. 6, 12, 14 for storing at 4C. <br />
</p><br />
</div></div><br />
<br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_11
Team:HokkaidoU Japan/Notebook/aggregation Week 11
2012-09-26T21:35:24Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==September 10th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 by 3piece ligation, we digested pBAD-RBS-eCFP-RBS-pSB1A2 by EcoRI and SpeI, Ag43-dT-pSB1AK3 (previously digested by HindIII) by XbaI and NotI, and pSTV28 by EcoRI and NotI. Then we digested pT7-RBS-pSB1C3 by XbaI and SpeI as a control for confirmation of the ability of restriction enzyme.<br />
<br /><br />
Insert1 (pBAD-RBS-eCFP-RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|17 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
Insert2 (Ag43-dT-pSB1AK3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|25 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xK buffer<br />
|1.5 ul<br />
|-<br />
|100xBSA<br />
|0.3ul<br />
|-<br />
|DW<br />
|1.5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (15 ng/ul)<br />
|9 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|10xBSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30~40 ng/ul)<br />
|10 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120910 digestion Insert1-pBAD-RBS-eCFP-RBS-pSB1A2 Insert2-Ag43-dT-pSB1AK3 Vector-ptet-RBS-eYFP-dT-pSTV28 Control-pT7-RBS-pSB1A2.jpg|thumb|digestion result]]<br />
</p><br />
<br />
==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120910 EthaPre Insert1-Insert2-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert1 DNA solution is 50 ng/ul, Insert2 DNA solution is 10~15 ng/ul and Vector DNA solution is 10 ng/ul.<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (10 ng/ul)<br />
|4 ul<br />
|-<br />
|Insert1 DNA (50 ng/ul)<br />
|2 ul<br />
|-<br />
|Insert2 DNA (10~15 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
Transformation ligation product into DH5&alpha;.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 14 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 11th==<br />
<div><br />
==Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2==<br />
<p><br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(pbad-f2 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
*We noticed that this step was actually 68.9 degree after reaction. <br />
<br />
We used N1 (DW only) and N2(pBAD-RBS-Ag43-dT-pSB1A2)as controls.<br />
Desired product is about 542bp.<br />
<br />
[[image:HokkaidoU2012 120911 colop pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
Because of mis-setting the PCR program, we failed to amplify desired DNA fragment so we retried the colony PCR in same reagent, correct PCR machine program.<br />
<br />
[[image:HokkaidoU2012 120912 colop2 pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed that the ligated DNA contains at least Ag43-dT-BioBrick_Suffix complex. We selected No. 2,4 for incubation for plasmid extraction and No. 5,6 for Aggregation check.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 12th==<br />
<div><br />
==Analysis nucleotide sequence==<br />
<p><br />
We analyzed the nucleotide sequence of pBAD-RBS-Ag43-dT on pSB1AK3 and pSB1C3.<br />
We used these 6 kinds of primers.<br />
100b up EX-F primer, pBAD-f1 primer, pBAD-f2 primer, Ag43-f1 primer, Ag43-f2 primer, Ag43-f3 primer, Ag43-f4 primer, 200b down PS-R primer<br />
<br />
</p><br />
<br />
==Aggregation check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
Aggregation check for No. 5,6 colonies selected by colony PCR at August 11th.<br />
#Prepared 5 ml LBA into glass tubes.<br />
#Re-suspended 2 colony mixture (No. 2 and No. 5 respectively). <br />
#Incubated at 37C for 18 hrs.<br />
</p><br />
<br />
==Digestion of RBS-phaB-pSB1A2 as Vector and RBS-eYFP-dT as Insert==<br />
<p><br />
To make a construct of RBS-phaB-RBS-eYFP-dT-pSB1A2, we digested RBS-phaB-pSB1A2 with SpeI & PstI, RBS-eYFP-dT with XbaI & PstI.<br />
<br /><br /><br />
<br />
Insert (RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|25 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|NotI<br />
|1 ul<br />
|-<br />
|10xK buffer<br />
|1.5 ul<br />
|-<br />
|100xBSA<br />
|0.3 ul<br />
|-<br />
|DW<br />
|1.5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS-phaB-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120912 Digestion Insert(RBS-eYFP-dT)-Vector(RBS-phaB-pSB1A2)-Control(pBAD-RBS-eCFP-RBS-pSB1A2).jpg|thumb|digestion result]]<br />
</p><br />
<br />
==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120912 Ethapre 20min Insert-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 40 ng/ul and Vector DNA solution is 30 ng/ul.<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS, Ag43-dT and pSTV28==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (40 ng/ul)<br />
|3 ul<br />
|-<br />
|Insert DNA (30 ng/ul)<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 13th==<br />
<div><br />
==Transformation of RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product into JM109.<br />
#Mixed 2 ul ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 14 hrs.<br />
</p><br />
<br />
==Aggregation Check of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28==<br />
<p><br />
#Mixed 25 ul Arabinose solution (20 %) to 5 ml LBC in glass tube.<br />
#Incubated for 24 hrs at 37C.<br />
<br />
<br />
Result: The construct has aggregated not forming large cluster but small dot cluster, and the supernatant has muddiness.<br />
</p><br />
<br />
==Colony PCR of RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(pbad-f2 primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R down primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|64.4<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|120<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaB-pSB1A2)as controls.<br />
Desired product is about 1800~2000bp.<br />
<br />
[[image:HokkaidoU2012 120913 colop RBS-phaB-RBS-eYFP-dT-pSB1A2 1,2,,,N1,N2.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.<br />
</p><br />
<br />
<br />
==Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 2 colony mixture (No. 1 and No. 2 respectively). <br />
#Incubated at 37C for 14 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 14th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.<br />
<br /><br /><br />
Insert (RBS-phaB-RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|21 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(RBS-phaC-RBS-phaA-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120914 Digestion Insert Vector.jpg|thumb|digestion result]]<br />
<br />
We suppose the Insert DNA (RBS-phaB-RBS-eYFP-dT) has contained Vector-dimer DNA as contamination. But desired DNA fragment (about 1600 bp) bond also appeared in the result of migration. We picked up the fragment and extracted from gel.<br />
</p><br />
<br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120914 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (50~60 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 13 hrs.<br />
<br />
We succeeded to transform the cells but noticed that we have used wrong DNA solution. We'll try this DNA synthesis again tomorrow.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 15th==<br />
<div><br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of RBS-phaC-RBS-phaA-RBS-phaB-RBS-dT-pSB1A2, we digested RBS-phaC-RBS-phaA with SpeI and PstI, RBS-phaB-RBS-eYFP-pSB1A2 with XbaI and PstI.<br />
<br /><br /><br />
Insert (RBS-phaB-RBS-eYFP-dT)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|21 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Vector(RBS-phaC-RBS-phaA-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|6 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|70<br />
|20<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120915 Digestion Insert-Vector.jpg|thumb|digestion result]]<br />
<br />
</p><br />
==Ethanol precipitation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120915 Ethapre Insert-Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 20 ng/ul and Vector DNA solution is about 50~60 ng/ul.<br />
</p><br />
<br />
==Ligation of RBS-phaB-RBS-eYFP-dT and RBS-phaC-RBS-phaA-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Vector DNA (50~60 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
Transformation of ligation product into JM109.<br />
#Mixed 2 ul RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 17 hrs.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 16th==<br />
<div><br />
==Digestion of pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 and pT7-RBS-pSB1C3==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3, we digested pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28 with EcoRI and SpeI, and pT7-RBS-pSB1C3 with EcoRI and SpeI. As a control, we digested pT7-RBS-pSB1C3 with only EcoRI to confirm the digestibility of This DNA (The digestibility with SpeI has confirmed in August 26th).<br />
<br /><br /><br />
Insert (pBAD-RBS-eCFP-RBS-Ag43-dT-pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 35ng/ul)<br />
|41 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|5 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
Vector(pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (about 30~40 ng/ul)<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120916 Digestion Insert Vector Control.jpg|thumb|digestion result]]<br />
<br />
We confirmed 4 bonds in the result of Inset DNA digestion. A Bond which shows the highest concentration and places about 8k ~ 10k bp area would be pBAD-RBS-eCFP-RBS-Ag43-pSTV28 construct. We considered about it bond whether remained from digestion, or at first formed dimer and separated by digestion. In either case, our target product has about 5200 bp and there is appropriate bond. We extracted the target bond from TBE gel and went next step.<br />
</p><br />
==Ethanol precipitation of pBAD-RBS-eCFP-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged in 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged in 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120916 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
<br />
</p><br />
<br />
==Ligation of pBAD-RBS-eCFP-RBS-Ag43-dT and pSB1C3==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Insert DNA <br />
|4 ul<br />
|-<br />
|Vector DNA<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
Transformation ligation product into DH5&alpha;.<br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-Ag43-dT--pSB1C3 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 13 hrs.<br />
</p><br />
<br />
==Colony PCR of RBS-phaC-RBs-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(phaA-1083bp-F primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|Reverse Primer(PS-R down primer: 10 ng/ul)<br />
|0.8 ul<br />
|-<br />
|DW<br />
|4.4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|180<br />
|-<br />
|5<br />
|72<br />
|120<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
We used N1 (DW only) and N2(RBS-phaC-RBS-phaA-RBS-phaB-dT-pSB1A2)as controls.<br />
Desired product is about 1800~2000bp.<br />
<br />
[[image:HokkaidoU2012 120916 coloP RBS-phaC-RBS-phaA-RBS-phaB-RBS-eYFP-dT-pSB1A2.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We noticed the ligated DNA contains phaB-something-SP sequence and the length is about 1600~1800bp, at least over 1000 bp. We selected No. 1,2 for incubation for plasmid extraction and No. 3,4 for stock at 4C.<br />
</p><br />
<br />
==Incubation of RBS-phaB-RBS-eYFP-dT-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 2 colony mixture (No. 1 and No. 2 respectively). <br />
#Incubated at 37C for 20 hrs.<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_10
Team:HokkaidoU Japan/Notebook/aggregation Week 10
2012-09-26T21:30:12Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==September 3rd==<br />
<div><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pBAD-RBS-Ag43-dT was successfully ligated with pSB1C3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|10 ul<br />
|-<br />
|Forward Primer(Ag43-f4)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-Reverse)<br />
|1 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.2<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (pBAD-RBS-Ag43-dT on pSB1Ak3) as controls.<br />
Desired product is about 600bp.<br />
</p><br />
[[image:HokkaidoU2012_120903_ag43s_psb1c3.jpg|thumb|Colony PCR result]]<br />
<br />
==Colony PCR of eCFP-RBS-pSB1A2==<br />
<p><br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-F primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (ptetR-RBS-eCFP-dT-pSB1A2) as controls.<br />
Desired product is about 776bp.<br />
<br />
[[image:HokkaidoU2012 120903 colop eCFP-RBS-pSB1A2 EX-F PS-R (2).jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We confirmed that about 70% of ligated DNA formed our desired construct. We selected No. 2 and 5 colony for incubation and store No. 7 and 8 colony mixture at 4C.<br />
</p><br />
<br />
==Incubation of eCFP-RBS-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 2 colony mixture (No. 2 and No. 5 respectively). <br />
#Incubated at 37C for 17 hrs.<br />
</p><br />
<br />
<br />
==Estimation of concentration of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
No. 2,5 means the colony number of colony PCR of eCFP-RBS-pSB1A2.<br />
<br />
<br />
[[image:HokkaidoU2012 120903 mini-prep eCFP-RBS-1A2No.2 sameNo.jpg|thumb|electrophoresis result]]<br />
<br />
<br />
We estimated the concentration of eCFP-RBS-pSB1A2 is 30 ng/ul and pBAD-RBS-pSB1A2 is 50 ng/ul.<br />
</p><br />
<br />
<br />
<br />
==Digestion of eCFP-RBS-pSB1A2 and pBAD-RBS-pSB1A2==<br />
<p><br />
To make a construct of pBAD-RBS-eCFP-RBS-pSB1A2, we digested eCFP-RBS-pSB1A2 with XbaI & PstI and pBAD-RBS-pSB1A2 with SpeI and PstI. And we digested eCFP-pSB1A2 with XbaI and SpeI as a control for confirmation of the ability to digest.<br />
<br />
Insert (eCFP-RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|22 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|3 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(pBAD-RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (50 ng/ul)<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (eCFP-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120903 Digesiton eCFP-RBS(XP) pBAD-RBS-pSB1A2(SP) Control(XS).jpg|thumb|digestion result]]<br />
<br />
<br />
From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 4th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pBAD-RBS-Ag43-dT on pSB1C3.<br />
We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 50 ul of elution buffer. <br />
<br />
</p><br />
[[image:HokkaidoU2012_120903_ag43_comple_psb1c3.jpg|thumb|Plasmid extraction result]]<br />
<br />
==Ethanol precipitation of digestion products (eCFP-RBS and pBAD-RBS-pSB1A2) and estimation of concentration==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120904 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
<br />
We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 50 ng/ul.<br />
</p><br />
<br />
==Ligation of eCFP-RBS and pBAD-RBS-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (50 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of pBAD-RBS-eCFP-RBS-pSB1A2==<br />
<p><br />
#Mixed 2 ul pBAD-RBS-eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 25 hours.<br />
</p><br />
<br />
<br />
==Digestion of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
To make a construct of ptet-RBS-eYFP-dT-pSTV28, we digested ptet-RBS-eYFP-dT-pSB1A2 with EcoRI and PstI, and pSTV28 with EcoRI and PstI. And we digested Ag43-dT-pSB1AK3 by HindIII.<br />
<br />
Insert (ptet-RBS-eYFP-dT-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(pSTV28)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (50 ng/ul)<br />
|3 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ag43-dT-pSB1AK3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|30 ul<br />
|-<br />
|HindIII<br />
|2 ul<br />
|-<br />
|10xM buffer<br />
|5 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
<br />
<br />
[[image:HokkaidoU2012 120906 Digestion Insert Vector Ag43-dT-1AK3(HindIII).jpg|thumb|digestion result]]<br />
<br />
<br />
We confirmed that ptet-RBS-eYFP-dT-pSB1A2 was partially digested and pSTV28 would be successfully digested we think. But we couldn't confirm whether Ag43-dT-pSB1AK3 was digested or not. <br />
We did a gel-extraction of these digested DNA and got 50ul solution of each DNA.<br />
</div></div><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 5th==<br />
<div><br />
==Digestion==<br />
<p><br />
We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) by restriction enzyme.<br />
Because I'd like to check the size of insert and vector of getting plasmid.<br />
<br />
<br />
No. 1<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
No. 2<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|10xBSA buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|6 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
No. 3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
No. 4<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
No. 5<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|2 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|14 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120905_ag43_comp_dig.jpg|thumb|Digestion resulsts]]<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==September 6th==<br />
<div><br />
==Ag43 expressing==<br />
<p><br />
We usually used plastic tube for incubating.<br />
<br />
The E. coli expressing Ag43 attachment to the face of tube wall.<br />
[[image:HokkaidoU2012_IMG_2100.jpg|thumb|plastic incubated results]]<br />
<br />
We estimated the reason is attachment to hydrophobic materials.<br />
We incubated in 1 ml of LBA (one contained 1% of L-arabinose, another did not contain L-arabinose) at 37C in glass tubes.<br />
</p><br />
<br />
==Ethanol precipitation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120906 Ethapre Insert Vector.jpg|thumb|ethanol precipitation result]]<br />
We confirmed that the concentration of Insert DNA solution is 10~20 ng/ul and Vector DNA solution is 30~40 ng/ul.<br />
</p><br />
<br />
==Ligation of ptet-RBS-eYFP-dT-pSB1A2 and pSTV28==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (30~40 ng/ul)<br />
|1.5 ul<br />
|-<br />
|Insert DNA (10~20 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|DW<br />
|0.5 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of ptet-RBS-eYFP-dT-pSTV28==<br />
<p><br />
#Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBC).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 19 hrs.<br />
</p><br />
<br />
<br />
==Colony PCR of pBAD-RBS-eCFP-RBS-pSB1A2==<br />
<p><br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(pbad-f2 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.3<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) as controls.<br />
Desired product is about 900bp.<br />
<br />
[[image:HokkaidoU2012 120906 colop pBAD-RBS-eCFP-RBS-pSB1A2.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We thought that almost all of ligated DNA successfully ligated to our desired construct. We selected No. 1 colony for incubation and store No. 2,3 and 4 colony liquid medium at 4C.<br />
</p> <br />
<br />
==Incubation of pBAD-RBS-eCFP-RBS-pSB1A2 for plasmid extraction==<br />
<p><br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 1 colony mixture. <br />
#Incubated at 37C for 18 hrs.<br />
</p><br />
<br />
==Transformation of ptet-RBS-eYFP-dT-pSTV28 and Test to confirm appropriate antibiotic amount for screening of pSTV28==<br />
<p><br />
To confirm how amount of chloramphenicol is needed for screening pSTV28 (Low copy plasmid) , we plated transformed cells on the special LBC which contains half amount of chloramphenicol we usually add. Now we call this LBC plate medium as LB1/2C.<br />
#Mixed 2 ul ptet-RBS-eYFP-dT-pSTV28 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LB1/2C).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 19 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 7th==<br />
<div><br />
==Estimation of concentration of pBAD-RBS-eCFP-RBS-pSB1A2==<br />
<p><br />
<br />
[[image:HokkaidoU2012 120907 mini-prep pBAD-RBS-eCFP-RBS-pSB1A2.jpg|thumb|electrophoresis result]]<br />
<br />
<br />
We estimated the concentration of pBAD-RBS-eCFP-RBS-pSB1A2 is about 30 ng/ul.<br />
</p><br />
<br />
==Incubation of ptet-RBS-eYFP-dT-pSTV28 for plasmid extraction==<br />
<p><br />
#Prepared 5~6 ml LBC into culture tubes.<br />
#Re-suspended 2 colony. <br />
#Incubated at 37C for 7 hrs.<br />
<br />
[[image:HokkaidoU2012 120907 mini-prep(Low-copy) ptet-RBS-eYFP-dT-pSTV28.jpg|thumb|Plasmid extraction result]]<br />
<br />
</p><br />
<br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_9
Team:HokkaidoU Japan/Notebook/aggregation Week 9
2012-09-26T20:32:31Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 27th==<br />
<div><br />
<br />
==Aggregation check==<br />
<p><br />
We found a lot of colonies on LBA plate, spreaded at August 26th.<br />
We incubated 3 colonies in LBAK solution including 1% L-arabinose for 18 hrs.<br />
However, we could not find expression of Ag43 protein.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
</p><br />
[[image:HokkaidoU2012_120827_take-iroiro.jpg|thumb|Plasmid extraction resulsts]]<br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120827 colop pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
The result did not show the band clearly. We selected No.1 and 2 colony for incubation.<br />
<br />
<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
Incubation of pT7-RBS-Ag43-dT on pSB1C3 in LBC liquid medium.<br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 2 colonies (No.1 and No.2 respectively). <br />
#Incubated at 37C for 15 hrs.<br />
</p><br />
<br />
==Transformation of ptet-RBS(B0034)-CFP-dT on pSB1A2==<br />
<p><br />
#Mixed 1 ul DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hours. <br />
</p><br />
<br />
==PCR of RBS-YFP-dT==<br />
<p><br />
Amplified the construct with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.2<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 28th==<br />
<div><br />
<br />
==Digestion==<br />
<p><br />
We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) by EcoRI and SpeI.<br />
Because I'd like to check the size of insert and vector of getting plasmid.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120828_pbad-rbs-ag43-dt-dig.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
<br />
==Plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT on pSB1C3. We re-suspended No.1,2 colonies and incubated.<br />
<br />
[[image:HokkaidoU2012 120828 mini-prep pT7-RBS-Ag43-dT No.jpg|thumb|Plasmid extraction result]]<br />
<br />
</p><br />
<br />
==Estimation of Concentration of RBS-eYFP-dT and ptetR-pSB1A2==<br />
<p><br />
{|<br />
|Solution<br />
|Value (ul)<br />
|-<br />
|1kb ladder<br />
|1<br />
|-<br />
|Insert<br />
|1<br />
|-<br />
|Vector<br />
|1<br />
|-<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120828 Vector-ptetonpSB1C3-Concentrationcheck Insert-rbs-yfp-dt-PCR.jpg|thumb|Electrophoresis result]]<br />
<br />
<br />
From this result, We estimated that the concentration of Insert DNA solution is about 40 ng/ul, and Vector DNA is also about 40 ng/ul.<br />
</p><br />
<br />
==Digestion of ptet-pSB1A2 and RBS-Ag43-dT==<br />
<p><br />
ptet-pSB1A2(Vector) was digested with SpeI and PstI, and RBS-Ag43-dT(Insert) was cut with XbaI and PstI.<br />
<br />
<br />
<br />
<br />
Vector<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|5 ul<br />
|-<br />
|SpeI<br />
|0.5 ul<br />
|-<br />
|PstI<br />
|0.5 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Inset<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|14 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120828 Digestion Insert-rbs-yfp-dt-PCR(XP) Vector-ptetonpSB1C3(SP).jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that Insert and Vector DNA were digested.<br />
</p><br />
<br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for ptet-pSB1A2 and RBS-eYFP-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
<br />
==Ligation==<br />
<p><br />
Ligation for ptet-pSB1A2 and RBS-eYFP-dT.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|3 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product (ptet-RBs-eYFP-dT on pSB1A2) into DH5&alpha;.<br />
#Mixed 1 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LBA.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hrs.<br />
</p><br />
<br />
<br />
==Gel extraction product check of pT7-RBS-Ag43-dT on pSB1C3==<br />
<p><br />
We couldn't get desired plasmid DNA by transformation. We doubted contamination of non-digested vector DNA and decided to test the gel extraction product of pT7-RBS on pSB1C3, which were successfully separated from non-digested product or not by transformation.<br />
#Mixed 1 ul of each DNA of ligation product and digestion product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol.<br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 16 hrs. <br />
<br />
<br />
There were no colony on the LBC plate that was spread solution mixed digestion product.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 29th==<br />
<div><br />
==Aggregation check==<br />
<p><br />
We incubated 9 colonies transformed at August 25th in LBAK solution including 1% L-arabinose for 16 hrs.<br />
Then we find that 1 culture expressing Ag43.<br />
Observed the E. coli cluster by a microscope of 100 magnifications.<br />
<br />
<br />
Then we checked whether pBAD promoter is behaving exactly or not.<br />
#1 colony ( could expressing Ag43 ) incubated in 2 ml LB at 37C for 2 hrs.<br />
#Prepared 5 kinds of LB medium differing the concentration of L-arabinose.<br />
<br />No. 1 :0.001%<br />
<br />No. 2 :0.1% <br />
<br />No. 3 :0.4%<br />
<br />No. 4 :1.0%<br />
<br />No. 5 :0%<br /><br />
#Added 400 ul of culture in several LB medium.<br />
#Incubated for 17 hrs and 30 min at 37C.<br />
<br />
</p><br />
<br />
==PCR of eCFP(E0020)==<br />
<p><br />
Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Desired product is about 800~900bp.,br />,br /><br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.2<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR of pT7-RBS-Ag43-dT on pSB1C3 transformed at August 28th. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp. This length is almost same as N2.<br />
<br />
[[image:HokkaidoU2012 120829 colop pT7-RBs-Ag43-dTonpSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
<br />
We decided to incubate the colony of No.7 and 8.<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of pT7-RBS-Ag43-dTonpSB1C3 and Ag43-dTonpSB1AK3==<br />
<p><br />
Incubation of pT7-RBS-Ag43-dTonpSB1C3 (and Ag43-dT on pSB1AK3) in LBC (LBA) liquid medium.<br />
#Prepared 2 ml LBC (LBA) into culture tubes.<br />
#Re-suspended 2 colonies No.7 and No.8 respectively. Ag43-dT on pSB1AK3 was re-suspended the N2 colony. <br />
#Incubated at 37C for 17 hrs.<br />
</p><br />
<br />
<br />
==Estimation of Concentration of RBS-eYFP-dT (PCR product) and ptetR-pSB1A2==<br />
<p><br />
{|<br />
|Solution<br />
|Value (ul)<br />
|-<br />
|1kb ladder<br />
|1<br />
|-<br />
|Insert<br />
|1<br />
|-<br />
|Vector<br />
|1<br />
|-<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120829 PCR 100bpup-eCFP-200bpdown mini-prep B0034.jpg|thumb|Electrophoresis result]]<br />
<br />
<br />
From this result, We estimated that the concentration of Insert DNA solution is about 36 ng/ul, and Vector DNA is also about 29 ng/ul.<br />
</p><br />
<br />
==Digestion of ptet-pSB1A2 and RBS-Ag43-dT==<br />
<p><br />
ptet-pSB1A2(Vector) was digested with EcoRI and SpeI, and RBS-Ag43-dT(Insert) was cut with EcoRI and XbaI.<br />
And we used construct of pT7-RBS on pSB1C3 as control for the function of XbaI.<br />
<br />
<br /><br /><br />
<br />
Insert (eCFP)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (36 ng/ul)<br />
|5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS on pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (29 ng/ul)<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBs on pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|100x BSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120829 Digestion eCFP RBSonpSB1A2 XbaIcontrol.jpg|thumb|digestion result]]<br />
<br />
From this result, vector DNA were digested by XbaI, but remains some amount of non-digested DNA.<br />
And insert DNA solution were not existed in both d- and d+ lines.It means we failed to extract from gel after electrophoresis. We decided to retry PCR.<br />
</p><br />
<br />
==PCR of eCFP(E0020)==<br />
<p><br />
Amplified the part with 100bp-up-EX primer and 200bp-down-PS primer.<br />
Desired product is about 800~900bp.<br /><br /><br />
Mixed PCR solutions.<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|Solution<br />
|Volume (ul)<br />
|-<br />
|DNA<br />
|1<br />
|-<br />
|100bp-up-EX<br />
|1<br />
|-<br />
|200bp-down-PS<br />
|1<br />
|-<br />
|MgSO4<br />
|3<br />
|-<br />
|dNTP<br />
|5<br />
|-<br />
|10x KOD-Plus-Neo Buffer<br />
|5<br />
|-<br />
|KOD-Plus-Neo<br />
|1<br />
|-<br />
|DW<br />
|33<br />
|-<br />
|Total<br />
|50<br />
|}<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58.0<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 30th==<br />
<div><br />
<br />
==Estimation of concentration of eCFP, pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT-pSB1AK3==<br />
<p><br />
No. 7,8 means the colony number of colony PCR of pT7-RBS-Ag43-dT-pSB1C3.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP mini-prep pT7-RBS-Ag43-dTonpSB1C3 No.jpg|thumb|electrophoresis result]]<br />
<br />
<br />
We failed the PCR for E0020.<br />
We estimated the concentration and the result was that pT7-RBS-Ag43-dT-pSB1C3 is 31 ng/ul and Ag43-dT-pSB1AK3 is 35 ng/ul.<br />
</p><br />
<br />
==Digestion of pT7-RBS-pSB1C3 and Ag43-dT-pSB1AK3==<br />
<p><br />
Digestion of pT7-RBS-pSB1C3 (as Vector) and Ag43-dT-pSB1AK3 (as Insert) to make pT7-RBs-Ag43-dT-pSB1C3 construct.<br />
<br /><br /><br />
Vector<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (31 ng/ul)<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xm buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|16 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Inset<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|14 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
As a background, we did PCR for eCFP again.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP digestion-Ag43-dTonpSB1AK3.jpg|thumb|digestion and PCR result]]<br />
<br />
<br />
We did not do digestion of vector DNA sokution, result, because it has only too low concentration to be digested product solution.<br />
<br />
<br /><br />
We failed PCR again.<br />
The digestion result was confirmed that the Insert DNA was successfully digested by XbaI and SpeI.<br />
And we extracted the insert DNA digestion product from agarose gel by gel-extraction kit and got 20 ul of Insert DNA solution. <br />
</p><br />
<br />
==Digestion of pSB1AK3 by hindIII==<br />
<p><br />
The gel-extracted DNA solution contains Ag43-dT DNA and pSB1AK3 DNA because these DNA have same size of base pair and exist as same band in agarose gel. Thus we digest pSB1AK3 with HindIII to separate it from Ag43-dT.<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|20 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|10<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
As a background, we did PCR for eCFP again.<br />
<br />
<br />
[[image:HokkaidoU2012 120830 PCR-eCFP digestion(HindIII)-Ag43-dTonpSB1AK3.jpg|thumb|PCR and digestion result]]<br />
<br />
<br />
We failed the PCR for E0020.<br />
The digestion result was confirmed that the insert DNA was successfully digested with HindIII.<br />
</p><br />
<br />
==Ethanol precipitation of pT7-RBS-Ag43-dT-pSB1C3 and Ag43-dT==<br />
<p><br />
We did ethanol precipitation to get more high concentration DNA solution of each part. And we got 5ul of each DNA solution.<br />
<br />
[[image:HokkaidoU2012 120831 Insert-d--d+ Vector-d+.jpg|thumb|ethanol precipitation result]]<br />
<br />
The electrophoresis result showed that the pT7-RBS-pSB1C3 construct was successfully digested by SpeI and get enough concentration of DNA solution to ligate. <br />
</p><br />
<br />
==Ligation of pT7-RBS-pSB1C3 and Ag43-dT==<br />
<p><br />
Ligation of pT7-RBS-pSB1C3 (as Vector) and Ag43-dT (as Insert).<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|2.5 ul<br />
|-<br />
|Insert DNA<br />
|2.5 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
[[image:HokkaidoU2012 120831 Ligation Vector Insert Ligationproduct(4ul).jpg|thumb|ligation result]]<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of Ag43-dT-pSB1AK3 and ptet-RBS-YFP-dT-pSB1A2==<br />
<p><br />
Incubation of Ag43-dT-pSB1AK3 and ptet-RBS-YFP-dT-pSB1A2 in LBA liquid medium.<br />
#Prepared 2 ml LBC into culture tubes.<br />
#Re-suspended 1 colony from each plate that each construct plated. <br />
#Incubated at 37C for 22 hrs (Ag43-dT-pSB1AK3 was incubated for 41 hrs)<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 31st==<br />
<div><br />
==Plasmid extraction for pBAD-RBS-Ag43-dT on pSB1AK3==<br />
<p><br />
We used promega SV miniprep kit.<br />
We got 100 ul DNA solution.<br />
</p><br />
[[image:HokkaidoU2012_120831_pbad-rbs-ag43-dt-on1ak3.jpg|thumb|plasmid extraction result]]<br />
<br />
==Digestion==<br />
<p><br />
We digested plasmid extraction products (pBAD-RBS-Ag43-dT on pSB1AK3) with EcoRI and SpeI.<br />
Because I'd like to check the size of insert and vector of getting plasmid.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120831_take-iroiro2.jpg|thumb|digestion result]]<br />
<br />
==Transformation of pT7-RBS-Ag43-dT-pSB1C3 and eCFP (E0020)==<br />
<p><br />
#Mixed 2 ul ligation product and 1ul E0020 DNA solution to each 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA and LBC).<br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol (E0020 mixture skipped this step).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 15 hrs.<br />
<br />
We failed to transformation of pT7-RBS-Ag43-dT-pSB1C3. <br />
</p><br />
<br />
==Confirmation of amplification of ptet-RBS-eYFP-dT-pSB1A2==<br />
<p><br />
After plasmid extraction, we confirmed whether the desired construct ptet-RBS-eYFP-dT was really amplified or not by PCR.<br />
We chose EX-F primer as forward primer and PS-R primer as reverse primer then amplified about 1000bp of insert DNA.<br />
As controls, we chose DW as N1 and ptet-RBS-eCFP-dT construct as N2.<br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-F primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|68.9<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
<br />
[[image:HokkaidoU2012 120831 colop N1(DW) N2(ptet-RBS-eCFP-dT) 1(ptet-RBS-eYFP-dT).jpg|thumb|PCR result]]<br />
<br />
<br />
This image showed that the desired construct (about 1000 bp) amplified by incubation.<br />
</p><br />
<br />
<br />
==Incubation for plasmid extraction of eCFP-pSB1A2==<br />
<p><br />
Incubation of eCFP-pSB1A2 in LBA liquid medium.<br />
#Prepared 2 ml LBA into culture tubes.<br />
#Re-suspended 1 colony. <br />
#Incubated at 37C for 16 hrs.<br />
</p><br />
<br />
==Transformation of ptet-RBS-eYFP-dT-pSB1A2 and pT7-RBS-Ag43-dT-pSB1C3==<br />
<p><br />
#Mixed 2 ul pT7-RBS-Ag43-dT-pSB1C3 ligation product and 1ul ptet-RBS-eYFP-dT-pSB1A2 DNA solution to each 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA and LBC).<br />
#Incubated for 2 hrs to get the resistance to Chloramphenicol (ptet-RBS-eYFP-dT-pSB1A2 mixture skipped this step).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 1st==<br />
<div><br />
==Digestion for pSB1C3==<br />
<p><br />
We have to change the plasmid backbone to pSB1C3.<br />
We got the insert DNA at August 31th.<br />
So we need to get the pSB1C3 solution digested with EcoRI and SpeI.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pSB1C3 (60 ng/ul)<br />
|3 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120902_take-iroiro.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of pBAD-RBS-Ag43-dT solution and pSB1C3 solution digested by EcoRI & SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for insert DNA and pSB1C3.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|1 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 1 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Plated 300 ul of the culture onto first LBC dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBC dish and spread.<br />
#Incubated the plates at 37C for 18 hrs. <br />
</p><br />
<br />
==Plasmid extraction of Ag43-dT-pSB1AK3 and RBS-pSB1A2==<br />
<p><br />
We used plasmid extraction kit of Nippon genetics and got 50 ul Ag43-dT-pSB1AK3 plasmid DNA solution.<br />
</p><br />
<br />
==Digestion of eCFP-pSB1A2 and RBS-pSB1A2==<br />
<p><br />
To make a construct of eCFP-RBS-pSB1A2, we digested eCFP-pSB1A2 by EcoRI and SpeI, and RBS-pSB1A2 with EcoRI and XbaI. And we digested pT7-RBS-pSB1C3 by XbaI as a control for confirmation of the ability to digest.<br />
<br /><br /><br />
Insert (eCFP-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (35 ng/ul)<br />
|11 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
<br />
Vector(RBS-pSB1A2)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (29 ng/ul)<br />
|6 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
control (pT7-RBS-pSB1C3)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (30 ng/ul)<br />
|6 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|100x BSA<br />
|0.2 ul<br />
|-<br />
|DW<br />
|11 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|120<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120902 Digestion I-+ V-+ C(XbaI)-+.jpg|thumb|digestion result]]<br />
<br />
<br />
From this image, we confirmed that DNA were digested into fragments and all of restriction enzyme worked. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==September 2nd==<br />
<div><br />
==Ethanol precipitation of digestion products (eCFP and RBS-pSB1A2) and estimation of concentration==<br />
<p><br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120902 Ethapre V I.jpg|thumb|ethanol precipitation result]]<br />
<br />
We estimated the concentration of ethanol presipitation products.The concentration of Insert DNA solution is about 20 ng/ul and Vector DNA solution is about 37 ng/ul.<br />
</p><br />
<br />
==Incubation of pT7-RBS-Ag43-dT-pSB1C3 transformation product==<br />
<p><br />
We failed to cultivation of above construct two times so we tried to incubation in LBC liquid medium.<br />
#Prepared 1.8 ml LBC into culture tubes.<br />
#Re-suspended 200 ul transformation product solution (which was mixed with LB and incubated for 3 hrs). <br />
#Incubated at 37C. <br />
</p><br />
<br />
==Ligation of eCFP and RBS-pSB1A2==<br />
<p><br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA (37 ng/ul)<br />
|1 ul<br />
|-<br />
|Insert DNA (20 ng/ul)<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|6 ul<br />
|-<br />
|Total<br />
|11 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
</p><br />
<br />
==Transformation of eCFP-RBS-pSB1A2==<br />
<p><br />
#Mixed 2 ul eCFP-RBS-pSB1A2 ligation product to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Mixed 350 ul of LB. <br />
#Prepared and Labeled two plastic plates with LB plate medium which contained appropriate antibiotics (LBA).<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Mixed 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 19 hrs.<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<br />
<div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_8
Team:HokkaidoU Japan/Notebook/aggregation Week 8
2012-09-26T19:46:45Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 20th==<br />
<div><br />
==Single colony isolation==<br />
<p><br />
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.<br />
#Picked up one colony.<br />
#Incubated on LBK (dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp up primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.2<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls.<br />
Desired product is about 300~400bp.<br />
<br />
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.<br />
</p><br />
<br />
<br />
==PCR==<br />
<p><br />
PCR of BBa_I13453 (pBAD only part, it is not contain araC,).<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==liquid culture==<br />
<p><br />
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBK (LBC) into culture tubes.<br />
#Resuspended 1 colonies. <br />
#Incubated the tubes at 37C for 16 hours (19 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 21st==<br />
<div><br />
==PCR==<br />
<p><br />
PCR of pBAD(containing araC)-RBS.<br />
And, we checked the plasmid which we refined by plasmid extraction at August 18th is pBAD-RBS on pSB1A3 or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-rbs_pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Aggregation check==<br />
<p><br />
we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK.<br />
We checked the construct by induction of L-arabinose after 16 hours incubate.<br />
<br />
#2 ml of liquid culture divided two culture. (made two 1 ml culture)<br />
#Added 1 ml LBK in one culture as a negative control.<br />
#Added 900 ul LBK and 100 ul 20% L-arabinose.<br />
#Incubated at 37C 130 rpm for 2 hrs and 30 min.<br />
#Placed tubes on the table at 30 min.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used Plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
<br />
[[image:HokkaidoU2012 120821 pT7-RBS on pSB1C3 ColonyNo. 4,5 mini-prep.jpg|thumb|Plasmid extraction result]]<br />
<br />
<br />
The concentration of 20 ul of plasmid extraction products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2. <br />
<br />
</p><br />
<br />
==liquid culture==<br />
<p><br />
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBA (LBC) into culture tubes.<br />
#Resuspended 2 colonies. <br />
#Incubated the tubes at 37C for 18 hours (16 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 22nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|plasmid extraction result]]<br />
<br />
<br />
<br />
One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy.<br />
We tried plasmid extraction to the latter, we got the 20 ul of DNA solution.<br />
And then, we did electrophoresis the plasmid extraction products and (pBAD-RBS and pBAD) gel extract products.<br />
<br />
[[image:HokkaidoU2012_120822_take1.jpg|thumb|electrophoresis result(1% agarose gel)]]<br />
[[image:HokkaidoU2012_120822_take2.jpg|thumb|electrophoresis result(2% agarose gel)]]<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR of pT7-RBS on pSB1C3.<br /><br />
<br />
We used 4 kinds of primer set.<br />
<br /><br />
1 : EX-F , PS-R primer<br /><br />
2 : EX-F , 200b down primer<br /><br />
3 : 100b up , PS-R primer<br /><br />
4 : 100b up , 200b down primer<br /><br />
The density of primer solutions is 10 uM.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer<br />
|1 ul<br />
|-<br />
|Reverse Primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 23rd==<br />
<div><br />
==Digestion==<br />
<p><br />
Digestion for making pBAD-RBS-Ag43-dT on pSB1AK3.<br />
<br />
Digestion of pBAD-RBS.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pBAD-RBS (100 ng/ul)<br />
|12 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Digestion of Ag43-dT on pSB1AK3.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT (120 ng/ul)<br />
|7 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120824_ag43-dt-dig2.jpg|thumb|Ag43-dT digestion result]]<br />
[[image:HokkaidoU2012_120824_pbad-rbs-dig.jpg|thumb|pBAD-RBS digestion result]]<br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
</p><br />
==Digestion==<br />
<p><br />
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 257ng/ul)<br />
|9 ul<br />
|-<br />
|HindIII(15 U/ul)<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion HindIII Ag43-dT with Ag43.jpg|thumb|digestion result]]<br />
<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not confirm how many pSB1AK3 were remained as non-digested products.<br />
<br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 24th==<br />
<div><br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI.<br />
<br /><br />
<br /><br />
Ag43-dT on pSB1AK3<br />
<br /><br /><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pBAD-RBS<br /><br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 20ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion SpeI pT7-RBS on pSB1C3.jpg|thumb|pT7-RBS on pSB1C3 digestion result]]<br />
About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.<br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|4 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 25th==<br />
<div><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product into DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and electrophoresis result image of ligation products, then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligated DNA solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs. <br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|600 (10 hours)<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 26th==<br />
<div><br />
<br />
===Ligation===<br />
<p><br />
Ligation of pBAD-RBS and Ag43-dT on pSB1AK3<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS<br />
|3 ul<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Plated 300 ul of the culture onto first LBA dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBA dish and spread.<br />
#Incubated the plates at 37C for 17 hrs and 30 min.<br />
</p><br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120826 Ethanol precipitation Ag43-dT and pT7-RBS on pSB1C3 d+.jpg|thumb|Ethanol precipitation result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|0.25 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|DW<br />
|1.75 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for 18 hrs. <br />
<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_8
Team:HokkaidoU Japan/Notebook/aggregation Week 8
2012-09-26T19:29:10Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 20th==<br />
<div><br />
==Single colony isolation==<br />
<p><br />
Single colony isolation of pBAD-RBS-Ag43-dT on pSB1AK3.<br />
#Picked up one colony.<br />
#Incubated on LBK(dt,RBS,T7) and LBC(pLacI-RBS-Ag43) for 20 hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7 and RBS was successfully ligated with pSB1C3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(100bp up primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.2<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (pBAD(containing araC)-RBS on pSB1A3) as controls.<br />
Desired product is about 300~400bp.<br />
<br />
[[image:HokkaidoU2012 120820 pT7-RBS on pSB1C3 colop.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that ligated DNA has 300 ~ 400 bp and the desired products would have 331bp if it were amplified by 100bp up primer and 200bp down primer. Thus we confirmed that pT7-RBS on pSB1C3 was successfully ligated without no.6,9,10 colonies, but these 3 solution were evaporated because of our mistake. We selected No.4 and 5 colony for liquid culture.<br />
</p><br />
<br />
<br />
==PCR==<br />
<p><br />
PCR of BBa_I13453 (pBAD only part, it is not contain araC,).<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==liquid culture==<br />
<p><br />
Liquid culture for 3 colonies of pBAD-RBS-Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No.4 and 5 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBK (LBC) into culture tubes.<br />
#Resuspended 1 colonies. <br />
#Incubated the tubes at 37C for 16 hours (19 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 21st==<br />
<div><br />
==PCR==<br />
<p><br />
PCR of pBAD(containing araC)-RBS.<br />
And, we checked the plasmid which we refined by plasmid extraction at August 18th is pBAD-RBS on pSB1A3 or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer: 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-R primer: 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012_120821_pbad-rbs_pcr.jpg|thumb|PCR result]]<br />
<br />
</p><br />
==Aggregation check==<br />
<p><br />
we cultured the E. coli, which transformed pBAD-RBS-Ag43-dT on pSB1AK3 in LBK.<br />
We checked the construct by induction of L-arabinose after 16 hours incubate.<br />
<br />
#2 ml of liquid culture divided two culture. (made two 1 ml culture)<br />
#Added 1 ml LBK in one culture as a negative control.<br />
#Added 900 ul LBK and 100 ul 20% L-arabinose.<br />
#Incubated at 37C 130 rpm for 2 hrs and 30 min.<br />
#Placed tubes on the table at 30 min.<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No. 4 and 5 selected by the result of colony PCR at August 20th. We used Plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
<br />
[[image:HokkaidoU2012 120821 pT7-RBS on pSB1C3 ColonyNo. 4,5 mini-prep.jpg|thumb|Plasmid extraction result]]<br />
<br />
<br />
The concentration of 20 ul of plasmid extraction products were too low to digestion or do something so we retry liquid culture of other number of colony solution: No. 1 and No. 2. <br />
<br />
</p><br />
<br />
==liquid culture==<br />
<p><br />
Liquid culture for Ag43-dT on pSB1AK3 (and pT7-RBS on pSB1C3 colony No. 1 and 2 which were selected from the results of colony PCR).<br />
#Added 2 ml of LBA (LBC) into culture tubes.<br />
#Resuspended 2 colonies. <br />
#Incubated the tubes at 37C for 18 hours (16 hours).<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 22nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Mni-prep of pT7-RBS on pSB1C3 of colony No.1 and 2 selected by the result of colony PCR at August 20th. We used plasmid extraction kit of Nippon genetics: FastGene Plasmid mini kit and finally we eluted the DNA with 20 ul of elution buffer. <br />
<br />
[[image:HokkaidoU2012 120822 pT7-RBS on pSB1C3 mini-prep No.jpg|thumb|plasmid extraction result]]<br />
<br />
<br />
<br />
One of Ag43-dT on pSB1AK3 culture did not get muddy. And another one is only a little muddy.<br />
We tried plasmid extraction to the latter, we got the 20 ul of DNA solution.<br />
And then, we did electrophoresis the plasmid extraction products and (pBAD-RBS and pBAD) gel extract products.<br />
<br />
[[image:HokkaidoU2012_120822_take1.jpg|thumb|electrophoresis result(1% agarose gel)]]<br />
[[image:HokkaidoU2012_120822_take2.jpg|thumb|electrophoresis result(2% agarose gel)]]<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR of pT7-RBS on pSB1C3.<br /><br />
<br />
We used 4 kinds of primer set.<br />
<br /><br />
1 : EX-F , PS-R primer<br /><br />
2 : EX-F , 200b down primer<br /><br />
3 : 100b up , PS-R primer<br /><br />
4 : 100b up , 200b down primer<br /><br />
The density of primer solutions is 10 uM.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer<br />
|1 ul<br />
|-<br />
|Reverse Primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|30<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 23rd==<br />
<div><br />
==Digestion==<br />
<p><br />
Digestion for making pBAD-RBS-Ag43-dT on pSB1AK3.<br />
<br />
Digestion of pBAD-RBS.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|pBAD-RBS (100 ng/ul)<br />
|12 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
Digestion of Ag43-dT on pSB1AK3.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Ag43-dT (120 ng/ul)<br />
|7 ul<br />
|-<br />
|Eco RI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
</p><br />
[[image:HokkaidoU2012_120824_ag43-dt-dig2.jpg|thumb|Ag43-dT digestion result]]<br />
[[image:HokkaidoU2012_120824_pbad-rbs-dig.jpg|thumb|pBAD-RBS digestion result]]<br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation to get more high concentration of Ag43-dT on pSB1AK3 solution digested by XbaI and SpeI.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
</p><br />
==Digestion==<br />
<p><br />
Digestion to divide Ag43-dT and pSB1AK3 which has same number of bp as Ag43-dT by digested by HindIII.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 257ng/ul)<br />
|9 ul<br />
|-<br />
|HindIII(15 U/ul)<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|70<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion HindIII Ag43-dT with Ag43.jpg|thumb|digestion result]]<br />
<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII, but it could not confirm how many pSB1AK3 were remained as non-digested products.<br />
<br />
</p><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 24th==<br />
<div><br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1C3 by SpeI, and Ag43-dT on pSB1AK3 digested by EcoRI and XbaI, pBAD-RBS digested by EcoRI and PstI.<br />
<br />
<br />
Ag43-dT on pSB1AK3<br />
<br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|9 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
EcoRI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 120ng/ul)<br />
|7 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pBAD-RBS<br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution ( 20ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|180<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
<br />
<br />
[[image:HokkaidoU2012 120824 digestion SpeI pT7-RBS on pSB1C3.jpg|thumb|pT7-RBS on pSB1C3 digestion result]]<br />
About pT7-RBS on pSB1C3, we successfully digested the plasmid DNA and converted it to linear DNA.<br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|4 ul<br />
|-<br />
|Insert DNA<br />
|4 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|10 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120825 Ligation pT7-RBS-Ag43-dT on psB1C3.jpg|thumb|Ligation result]]<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 25th==<br />
<div><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the pT7-RBS-Ag43-dT on pSB1C3 was successfully ligated or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(200bp down primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53.0<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 695bp.<br />
<br />
[[image:HokkaidoU2012 120825 coloP pT7-RBS-Ag43-dT on pSB1C3.jpg|thumb|Colony PCR result]]<br />
<br />
The results showed that desired DNA were not existed in these picked up colonies. We observed our ethanol precipitation and ligation products electrophoresis result image then noticed that the concentration of each ethanol precipitation product was reversed. This means vector DNA would be self-ligated by the high ratio of molar in ligation reaction solution. We decided to try the DNA synthesis from digestion reaction of vector DNA once more time.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digest vector DNA: pT7-RBS on pSB1C3 by SpeI. Not to leave the plasmid DNA as plasmid DNA, we digested the DNA for 18 hrs. <br />
<br />
pT7-RBS on pSB1C3 (SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (20 ng/ul)<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Number<br />
|Degree<br />
|Minute<br />
|-<br />
|1<br />
|37<br />
|600 (10 hours)<br />
|-<br />
|2<br />
|60<br />
|15<br />
|-<br />
|3<br />
|4<br />
|HOLD<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 26th==<br />
<div><br />
<br />
===Ligation===<br />
<p><br />
Ligation of pBAD-RBS and Ag43-dT on pSB1AK3<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS<br />
|3 ul<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
===Transformation===<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Plated 300 ul of the culture onto first LBA dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second LBA dish and spread.<br />
#Incubated the plates at 37C for 17 hrs and 30 min.<br />
</p><br />
<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.<br />
<br />
</p><br />
<br />
==Ethanol Precipitation==<br />
<p><br />
Ethanol precipitation for pT7-RBS on pSB1C3 and Ag43-dT digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and air drying at room temperature then added 5 ul of DW. <br />
<br />
<br />
[[image:HokkaidoU2012 120826 Ethanol precipitation Ag43-dT and pT7-RBS on pSB1C3 d+.jpg|thumb|Ethanol precipitation result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for Ag43-dT as insert and pT7-RBS on pSB1C3 as vector.<br />
We use Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer. <br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Vector DNA<br />
|0.25 ul<br />
|-<br />
|Insert DNA<br />
|3 ul<br />
|-<br />
|DW<br />
|1.75 ul<br />
|-<br />
|Ligation Mighty Mix<br />
|5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was in detail below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation for ligation product in DH5&alpha;.<br />
#Added 2 ul of DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB. <br />
#Incubated the cells for 2 hours at 37C. <br />
#Prepared and Labeled two plastic plates with LBC.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread.<br />
#Incubated the plates at 37C for hours. <br />
<br />
</p><br />
</div></div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_7
Team:HokkaidoU Japan/Notebook/aggregation Week 7
2012-09-26T19:18:11Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 16th==<br />
<div><br />
==ligation==<br />
<p><br />
We ligated pBad-RBS on pSB1A3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS (5 ng/ul)<br />
|1 ul<br />
|-<br />
|Ag43-dT (25 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBA.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Ag43-dT on pSB1AK3 digestion with SpeI and XbaI.<br />
<br />
Ag43-dT<br />
SpeI and XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (100 ng/ul)<br />
|12 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
[[image:HokaidoU2012_120813_pbad-rbs-ori-x10.jpg|thumb|pBad-RBS on pSB1A3 electrophoresis result]]<br />
[[image:HokkaidoU2012_120813_ag43-dt-s-x.jpg|thumb|digestion result]]<br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 17th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pBAD-RBS on pSB1A3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1 colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer (50 pmol/ul))<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dt on pSB1K3) as controls.<br />
Desired product is about 800~1000bp.<br />
<br />
[[image:HokkaidoU2012_120817_pbad-rbs-ag43-dt-coloP.jpg|thumb|Colony PCR result]]<br />
<br />
From this result, N2 has about 500bp band.<br />
We use to liquid culture, No. 7,10,12.<br /><br />
We cultured these DNA in E. coli solution, after added 200 ul LB, at 37C for 3 hrs.<br />
Next step, we resuspended these 5 colonies and cultured (add 1800 ul LB and 2 ul Amp) for hours at 37C.<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of BBa_I13453 (pBAD on pSB1A3) in DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 350 ul of LB.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 19 hours.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
From sequencing results, we found that we failed to make pT7-RBS on pSB1K3.<br /><br />
So, we tried to make it again. <br /><br />
When the restriction enzyme digest the DNA, it is important for 3A assembry to be digested the target plasmid completely. So, we activated the restriction enzymes for 10 hours.<br /><br />
<br />
pT7 (BBa_I719005)<br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
RBS (BBa_B0034)<br />
XbaI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (40 ng/ul)<br />
|12.5 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
pSB1C3<br />
EcoRI/PstI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution (25 ng/ul)<br />
|12 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|PstI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 18th==<br />
<div><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for three digestion products.<br />
#Added 2 ul of NaoAc, 1.5 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 14000 rpm, 30 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Before digestion of pBAD-RBS on pSB1A3 and Ag43-dT on pSB1AK3, it is necessary to refine these two DNA. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 20 ul of DNA solution from 10 ul of plasmid extraction products.<br />
</p><br />
==Digestion==<br />
<p><br />
Digested pBAD-RBS on pSB1A3 as insert and Ag43-dT on pSB1AK3 as vector with EcoRI and SpeI to make a constract, pBAD-RBS-Ag43-dT on pSB1AK3.<br><br />
<br><br />
<br />
pBAD-RBS<br><br />
EcoRI/SpeI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|plasmid extraction product (20 ng/ul)<br />
|20 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
<br />
Ag43-dT on pSB1AK3<br><br />
EcoRI/XbaI<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|Gel extraction product (40 ng/ul)<br />
|8 ul<br />
|-<br />
|EcoRI<br />
|1 ul<br />
|-<br />
|XbaI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|3 ul<br />
|-<br />
|DW<br />
|17 ul<br />
|-<br />
|Total<br />
|30 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012_120818_pbad-rbs--ag43-dt-dig.jpg|thumb|Digestion result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion products.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction for colony number of No. 10 and 12 of pBAD-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions. <br />
[[image:HokkaidoU2012_120818_pbad-rbs-ag43-dt.jpg|thumb|plasmid extraction result]]<br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation for pT7 and RBS as inserts and pSB1C3 as vector.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7 (100 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT (100 ng/ul)<br />
|2 ul<br />
|-<br />
|pSB1C3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pBAD-RBS (80 ng/ul)<br />
|2 ul<br />
|-<br />
|Ag43-dT on pSB1AK3 (60 ng/ul)<br />
|2 ul<br />
|-<br />
|DW<br />
|1 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 19th==<br />
<div><br />
==Transformation==<br />
<p><br />
Transformation of ligation product (two construct ligated at August 18) in DH5&alpha;.<br /><br />
<br />
<br />
pT7-RBS on pSB1C3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LB.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBC.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 18 hours.<br />
<br />
pBAD-RBS-Ag43-dT on pSB1AK3<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 350 ul of LBK.<br />
#Incubated at 37C for 2 hours.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 300 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and plated 300 ul of it onto second dish and spread. <br />
#Incubated the plates at 37C for 16 hours and 30 minutes.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR for pT7-RBS on pSB1K3.<br />
By sequencing reaction, we found the No. 10 plasmid is not pT7-RBS on pSB1K3.<br />
We checked the other plasmid is the same or not.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Forward Primer(EX-Forward primer : 10 uM)<br />
|1 ul<br />
|-<br />
|Reverse Primer(PS-Reverse primer : 10 uM)<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|68<br />
|30<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~3 x 45<br />
<br />
[[image:HokkaidoU2012_120819_pcr.jpg|thumb|PCR result]]<br />
<br />
No. 4 plasmid has independent bands.<br />
However, if it is pT7-RBS on pSB1K3, it has about 60~70bp band.<br />
<br />
</p><br />
<br />
</div><div><br />
<br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_6
Team:HokkaidoU Japan/Notebook/aggregation Week 6
2012-09-26T19:08:52Z
<p>Kwt: </p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<div class="hokkaidou-notebook-daily"><br />
==August 6th==<br />
<div><br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|1 ul<br />
|-<br />
|Ag43-dT<br />
|2 ul<br />
|-<br />
|DW<br />
|2 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pBad-RBS. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012_120806_pBad-rbs.jpg|thumb|Plasmid extraction result]]<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA (pT7-RBS-Ag43-dT on pSB1K3) in DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 600 ul of LB then incubated the cells for 2 hours at 37C.<br />
#Prepared and Labeled two LBK plates.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 900 ul of LB to 100 ul of the culture and plated 300 ul of it onto second dish and spread. <br />
#Incubated them at 37C for 16 hours.<br />
<br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for plasmid extraction product (pBad-RBS). Because the refine of plasmid extraction product is not enough. <br /><br />
We used 15 ul of all solution.<br />
#Added 1.5 ul of NaoAc(3 M), 1.5 ul of glycogen and 38 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of ethanol precipitation product(pBad-RBS).<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
</p><br />
[[image:HokkaidoU2012_120807_pbad-rbs-digested.jpg|thumb|digestion result]]<br />
<br />
From this result, speI digested DNA completely.<br /><br />
So I think that the pT7-RBS on pSB1K3 DNA solution is not pure.<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 7th==<br />
<div><br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220 ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
</p><br />
<br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43 on pSB1C3 (K346007)) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU2012_120807_colonyPCR.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies of No. 1 and 2 and 5 and 11 and 14 have deep band.<br />
Next step, we resuspended these 5 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 15 hours at 37C.<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 8th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of colony No. 1 and 14. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012_120808_pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]<br />
<br />
And then, we did plasmid extraction for colony No. 2,5,11. The results of them are the same.<br />
</p><br />
<br />
==Sequencing==<br />
<p><br />
PCR for sequencing.<br />
<br />
{|<br />
|DNA<br />
|primer<br />
|-<br />
|Ag43 plasmid extraction product<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|K542009<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|-<br />
|pT7-RBS on pSB1C3<br />
|100bp-up forward primer<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|Ag43-dT on pSB1T3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|}<br />
<br />
<br />
Sequencing PCR<br />
{|class="hokkaidou-table-sequencing-pcr-reagent"<br />
|-<br />
|template DNA<br />
|1 ul<br />
|-<br />
|Ready Reaction Premix<br />
|1 ul<br />
|-<br />
|5x Sequencing Buffer<br />
|1.5 ul<br />
|-<br />
|H2O<br />
|5 ul<br />
|-<br />
|Primer(1 pmol/ul)<br />
|1.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|96<br />
|10<br />
|-<br />
|2<br />
|50<br />
|5<br />
|-<br />
|3<br />
|60<br />
|240<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:1~3 x 25<br />
<br />
<br />
To purify the PCR product, we did ethanol precipitation.<br />
<br />
Ethanol precipitation for sequencing.<br />
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 26C.<br />
#Removed supernatant and added 100ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 26C.<br />
#Removed supernatant and dried out at room temperature, after that added 10 ul of DW. <br />
<br />
<br />
Then ran a sequencing machine (ByoDye Terminator v3.1 Cycle Sequencing Kit)<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 9th==<br />
<div><br />
==Ligation==<br />
<p><br />
We ligated pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br /><br />
We did ligation (pT7-RBS on pSB1K3 and Ag43-dT) and transformation it, at August 6th.<br />
However, we could not get the target plasmid. (pT7-RBS-Ag43-dT on pSB1K3)<br />
So, we did ligation by using more Insert DNA part.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|1 ul<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation of plasmid DNA ligation product (pT7-RBS-Ag43-dT on pSB1K3) in DH5&alpha;.<br /><br />
Transformation of August 6th, we found only 14 colonies. So this time, we use 3 ul of DNA solution for transformation.<br />
<br />
#Added 3 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 400 ul of LB then incubated the cells for 2 hours at 37C.<br />
#Prepared and Labeled two LBK plates.<br />
#Plated 300 ul of the culture onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the culture and plated 300 ul of it onto second dish and spread. <br />
#Incubated them at 37C for 16 hours.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 11st==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul (1colony/10 ul DW)<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|53<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU1120811 coloP.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies of No. 6 and 8 is like N2 band.<br />
Next step, we resuspended these 2 colonies and cultured (add 1700 ul LB and 2 ul Amp) for 16 hours at 37C.<br />
<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 12nd==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of No. 6 and 8. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012_120812_pt7-dt.jpg|thumb|plasmid extraction result]]<br />
</p><br />
<br />
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{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project
Team:HokkaidoU Japan/Project
2012-09-26T18:55:41Z
<p>Kwt: </p>
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</style><br />
<h2>Project</h2><br />
<div class="hokkaidou-section"><br />
<a href="/Team:HokkaidoU_Japan/Project/Description" class="hokkaidou-project-index" id="hokkaidou-project-about"><br />
<h5>About this project</h5><br />
<p>hogehogehogehogehogehogehogehogehogehogehogehogehogehogehoeghoeghoeghoegho</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/Aggregation" class="hokkaidou-project-index" id="hokkaidou-project-aggregation"><br />
<h5>Aggregation Module</h5><br />
<p>You have no need to centrifuge anymore.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/PHB_Synthesis" class="hokkaidou-project-index" id="hokkaidou-project-phb"><br />
<h5>PHB Synthesis</h5><br />
<p>PHB has possibility to be utilized in many fields.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/Biocapsule" class="hokkaidou-project-index" id="hokkaidou-project-capsule"><br />
<h5>Biocapsule</h5><br />
<p>What happened when work these two modules?</p><br />
</a><br />
</div><br />
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Kwt
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Team:HokkaidoU Japan/Project
2012-09-26T18:50:24Z
<p>Kwt: </p>
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<h2>Project</h2><br />
<div class="hokkaidou-section"><br />
<a href="/Team:HokkaidoU_Japan/Project/Description" class="hokkaidou-project-index" id="hokkaidou-project-about"><br />
<h5>About this project</h5><br />
<p>hogehogehogehogehogehogehogehogehogehogehogehogehogehogehoeghoeghoeghoegho</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/Aggregation" class="hokkaidou-project-index" id="hokkaidou-project-aggregation"><br />
<h5>Aggregation Module</h5><br />
<p>You have no need to centrifuge anymore.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/PHB_Synthesis" class="hokkaidou-project-index" id="hokkaidou-project-phb"><br />
<h5>PHB Synthesis</h5><br />
<p>PHB will save the fossil fuels and has possibility to be utilized in many fields.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/Biocapsule" class="hokkaidou-project-index" id="hokkaidou-project-capsule"><br />
<h5>Biocapsule</h5><br />
<p>hogehogehogehogehogehogehogehogehogehogehogehogehogehogehoeghoeghoeghoegho</p><br />
</a><br />
</div><br />
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<br />
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{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Project
Team:HokkaidoU Japan/Project
2012-09-26T18:46:16Z
<p>Kwt: </p>
<hr />
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<h2>Project</h2><br />
<div class="hokkaidou-section"><br />
<a href="/Team:HokkaidoU_Japan/Project/Description" class="hokkaidou-project-index" id="hokkaidou-project-about"><br />
<h5>About this project</h5><br />
<p>hogehogehogehogehogehogehogehogehogehogehogehogehogehogehoeghoeghoeghoegho</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/Aggregation" class="hokkaidou-project-index" id="hokkaidou-project-aggregation"><br />
<h5>Aggregation Module</h5><br />
<p>You have no need to centrifuge anymore.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/PHB_Synthesis" class="hokkaidou-project-index" id="hokkaidou-project-phb"><br />
<h5>PHB Synthesis</h5><br />
<p>This will save the fossil fuels and has more possibility to be utilized.</p><br />
</a><br />
<a href="/Team:HokkaidoU_Japan/Project/Biocapsule" class="hokkaidou-project-index" id="hokkaidou-project-capsule"><br />
<h5>Biocapsule</h5><br />
<p>hogehogehogehogehogehogehogehogehogehogehogehogehogehogehoeghoeghoeghoegho</p><br />
</a><br />
</div><br />
</html><br />
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Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_5
Team:HokkaidoU Japan/Notebook/aggregation Week 5
2012-09-26T18:38:41Z
<p>Kwt: /* Digestion */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 30th==<br />
<div><br />
==digestion==<br />
<p><br />
I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.<br />
[[image:HokkaidoU2012_120730_ag43-f1-e-s.jpg|thumb|digestion result]]<br />
<br />
</p><br />
==Transformation==<br />
<p><br />
I think that the E. coli which we used transformation is BL21(DE3)pLysS.<br />
So, the E. coli could grow on LBC plate.<br /><br />
I'm going to do transformation , use DH5&alpha;.<br />
<br />
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 200 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish. <br />
#Incubated the plates at 37C for 14 hrs.<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 31st==<br />
<div><br />
==Liquid culture==<br />
<p><br />
Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended colonies.<br />
#Incubated the tubes at 37C for 18 hrs.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 1st==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT which had been incubated from 31st July. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012 120801 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br />
<br><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''pT7-RBS(30 ng/ul) '''=No. 10<br />
<br><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012 120801 pt7-rbs-digS.jpg|thumb|digestion result]]<br />
<br />
We couldn't digested them exactly, so we tried to digest once more time.<br><br />
<br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br><br />
SpeI 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture for pBAD-RBS on pSB1K3.<br />
#Added 2 ml of LBK into culture tube.<br />
#Scraped the surface of glycerol stock of construct.<br />
#Incubated the tube at 33C.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 2nd==<br />
<div><br />
<p><br />
==Preparing chemical competent cell==<br />
Preparing chemical competent cell of BL21, JM109 and DH5&alpha;.<br />
Chemical competent cell made in each E. coli strains.<br />
<br><br><br />
Our competent cell Protocol<br />
#Did single colony isolation on LB plate.<br />
#Incubated the plate for 15-19 hours at 37C.<br />
#Lifted colony of E. coli into 2 ml LB.<br />
#Incubated at 37C for 12-16 hrs at 180-200 rpm.<br />
#Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.<br />
#Incubated cells at 20C (over 24 hrs) at 140 rpm.<br />
#Selected culture by measuring OD 600.<br />
#Incubated the 300 ml flask for 10 min on ice.<br />
#Transfered the culture into two 50 ml Falcon tubes.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)<br />
#Collected them to one tube.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 3.2 ml of TB.<br />
#Instilled 0.24 ml of DMSO in precipitant.<br />
#Incubated the 50 ml Falcon tube for 10 min on ice.<br />
#Divided 50 ul of solutions in each 0.5 ml tubes.<br />
#Freezed the suspension by liquid nitrogen.<br />
#Stored at –80C.<br />
<br />
<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)<br />
<br />
[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|Electrophoresis result]]<br />
There were low concentration band above thick band. This thick band was same as digestion minus band.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 3rd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for the result of digestion.<br />
[[image:HokkaidoU2012 120803 pt7-rbs-dspe1.jpg|thumb|Electrophoresis result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation of pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Spread 300 ul of the transformation onto first dish.<br />
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish. <br />
#Incubated the plates at 37C for 15 hrs 45 min.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm Ag43-f4 primer.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-f4 primer<br />
|1 ul<br />
|-<br />
|PS-R primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120803 Ag43 PCR(Forward=ag43-f4 Reverse=PS-R).jpg|thumb|PCR result]]<br />
<br />
From this result, we could see that the ag43-f4 primer had worked and DNA of last 500~600 bp of ag43 to BioBrick suffix were increased.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 4th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU2012 120804 pt7-rbs-ag43-dt coloP.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band.<br />
Next step, we resuspended these three colonies and incubated. <br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture of colonies passed the colony PCR test.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended 3 colonies.<br />
#Incubated the tubes at 37C for 13 hrs. <br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 5th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
<br />
[[image:HokkaidoU2012 120805 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI.<br />
<br />
<br />
pT7-RBS on pSB1K3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS (once digestioned with SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 pT7-RBS digestion SpeI (no.jpg|thumb|digestion result]]<br />
From this result, the bottom band was digested incorrectly and middle band was digested correctly, we thought. Was the above band something contaminated in the DNA solution?<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction of middle band. We thought this band would be the exactly digested DNA fragment. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
==Sequencing==<br />
<p><br />
Sequencing to confirm what kind of DNA we made.<br />
<br />
<br />
{|<br />
|DNA<br />
|primer<br />
|-<br />
|Ag43 plasmid extraction product<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|K542009<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|-<br />
|pT7-RBS on pSB1C3<br />
|100bp-up forward primer<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|Ag43-dT on pSB1T3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|}<br />
<br />
<br />
Sequencing PCR<br />
{|class="hokkaidou-table-sequencing-pcr-reagent"<br />
|-<br />
|template DNA<br />
|1 ul<br />
|-<br />
|Ready Reaction Premix<br />
|1 ul<br />
|-<br />
|5x Sequencing Buffer<br />
|1.5 ul<br />
|-<br />
|H2O<br />
|5 ul<br />
|-<br />
|Primer(1 pmol/ul)<br />
|1.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|96<br />
|10<br />
|-<br />
|2<br />
|50<br />
|5<br />
|-<br />
|3<br />
|60<br />
|240<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:1~3 x 25<br />
<br />
<br />
To extract the PCR product, we did ethanol precipitation.<br />
<br><br />
Ethanol precipitation for sequencing.<br />
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 26C.<br />
#Removed supernatant and added 100ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 26C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of Hi-Di. <br />
<br />
<br />
Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)<br><br />
We could not get the sequencing data.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digesting of Ag43-dT (digested by SpeI and XbaI) with HindIII.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 Ag43-dT on pSB1AK3(d+ with XbaI &amp; SpeI) digestion with HindIII.jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
{{Team:HokkaidoU_Japan/footer}}</div>
Kwt
http://2012.igem.org/Team:HokkaidoU_Japan/Notebook/aggregation_Week_5
Team:HokkaidoU Japan/Notebook/aggregation Week 5
2012-09-26T18:37:24Z
<p>Kwt: /* Sequencing */</p>
<hr />
<div>{{Team:HokkaidoU_Japan/header}}<br />
{{Team:HokkaidoU_Japan/nav.notebook}}<br />
<div id="hokkaidou-column-main"><br />
<!-- DO NOT EDIT OVER THIS LINE @iTakeshi --><br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==July 30th==<br />
<div><br />
==digestion==<br />
<p><br />
I tried to digest the plasmid extraction products again, and after Ethanol precipitation, we did electrophoresis.<br />
[[image:HokkaidoU2012_120730_ag43-f1-e-s.jpg|thumb|digestion result]]<br />
<br />
</p><br />
==Transformation==<br />
<p><br />
I think that the E. coli which we used transformation is BL21(DE3)pLysS.<br />
So, the E. coli could grow on LBC plate.<br /><br />
I'm going to do transformation , use DH5&alpha;.<br />
<br />
Transformation plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30 min.<br />
#Added 200 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Plate 200 ul of the transformation onto first dish and spread.<br />
#Added 450 ul of LB to 50 ul of the transformation and spread 200 ul of it onto second dish. <br />
#Incubated the plates at 37C for 14 hrs.<br />
<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==July 31st==<br />
<div><br />
==Liquid culture==<br />
<p><br />
Liquid culture for pT7-RBS-Ag43-dT on pSB1K3.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended colonies.<br />
#Incubated the tubes at 37C for 18 hrs.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 1st==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of pT7-RBS-Ag43-dT which had been incubated from 31st July. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
[[image:HokkaidoU2012 120801 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction result]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br />
<br><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|3.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
'''pT7-RBS(30 ng/ul) '''=No. 10<br />
<br><br />
SpeI using 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xH buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
SpeI using 10xM<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|3 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
[[image:HokkaidoU2012 120801 pt7-rbs-digS.jpg|thumb|digestion result]]<br />
<br />
We couldn't digested them exactly, so we tried to digest once more time.<br><br />
<br />
'''pT7-RBS(20 ng/ul) '''=No. 9<br><br />
SpeI 10xH <br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4.5 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|12.5 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture for pBAD-RBS on pSB1K3.<br />
#Added 2 ml of LBK into culture tube.<br />
#Scraped the surface of glycerol stock of construct.<br />
#Incubated the tube at 33C.<br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 2nd==<br />
<div><br />
<p><br />
==Preparing chemical competent cell==<br />
Preparing chemical competent cell of BL21, JM109 and DH5&alpha;.<br />
Chemical competent cell made in each E. coli strains.<br />
<br><br><br />
Our competent cell Protocol<br />
#Did single colony isolation on LB plate.<br />
#Incubated the plate for 15-19 hours at 37C.<br />
#Lifted colony of E. coli into 2 ml LB.<br />
#Incubated at 37C for 12-16 hrs at 180-200 rpm.<br />
#Transfered 30 ul, 100 ul, 300 ul of the culture into 100 ml of LB.<br />
#Incubated cells at 20C (over 24 hrs) at 140 rpm.<br />
#Selected culture by measuring OD 600.<br />
#Incubated the 300 ml flask for 10 min on ice.<br />
#Transfered the culture into two 50 ml Falcon tubes.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 15 ml of TB (Transformation Buffer).(7.5 ml/tube)<br />
#Collected them to one tube.<br />
#Centrifuged at 3000 rpm, 4C for 20 min (TOMY LX-120 rotor), and discard supernatant.<br />
#Suspended the pellet in ice-cold 3.2 ml of TB.<br />
#Instilled 0.24 ml of DMSO in precipitant.<br />
#Incubated the 50 ml Falcon tube for 10 min on ice.<br />
#Divided 50 ul of solutions in each 0.5 ml tubes.<br />
#Freezed the suspension by liquid nitrogen.<br />
#Stored at –80C.<br />
<br />
<br />
</p><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis of digestion result of August 1st.(pT7-RBS on pSB1K3 digested by SpeI)<br />
<br />
[[image:HokkaidoU2012 120802 pT7-RBS on pSB1K3 SpeI.jpg|thumb|Electrophoresis result]]<br />
There were low concentration band above thick band. This thick band was same as digestion minus band.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digestion of pT7-RBS on pSB1K3 (more fresh one) with SpeI.<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
</p><br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
<br />
==August 3rd==<br />
<div><br />
<br />
==Electrophoresis==<br />
<p><br />
Electrophoresis for the result of digestion.<br />
[[image:HokkaidoU2012 120803 pt7-rbs-dspe1.jpg|thumb|Electrophoresis result]]<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion product. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
<br />
==Ethanol precipitation==<br />
<p><br />
Ethanol precipitation for digestion and gel extraction product.<br />
#Added 5 ul of NaoAc, 1.5 ul of glycogen and 125ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 10 min at 4C.<br />
#Removed supernatant and added 220ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 4C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of DW. <br />
</p><br />
<br />
==Ligation==<br />
<p><br />
Ligation of pT7-RBS on pSB1K3 (digested Spe1) as vector and Ag43-dT (digested Spe1 and Xba1 and HindIII) as insert.<br />
<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|pT7-RBS<br />
|2 ul<br />
|-<br />
|Ag43-dT<br />
|4 ul<br />
|-<br />
|Ligation Mighty Mix(TAKARA)<br />
|6 ul<br />
|-<br />
|Total<br />
|12 ul<br />
|}<br />
<br />
<br />
Ligation reaction time was written below.<br />
<br />
{|class="hokkaidou-table-ligation"<br />
|-<br />
|Degree<br />
|Minute<br />
|-<br />
|16<br />
|30<br />
|-<br />
|65<br />
|10<br />
|-<br />
|4<br />
|Hold<br />
|}<br />
</p><br />
<br />
==Transformation==<br />
<p><br />
Transformation plasmid DNA ligated product (pT7-RBS-Ag43-dT on pSB1K3) into DH5&alpha;.<br />
#Added 1 ul of plasmid DNA to 50 ul of thawed competent cells on ice.<br />
#Incubated on ice for 30min.<br />
#Added 600 ul of LB then incubated the cells for 2 hrs at 37C.<br />
#Prepared and Labeled two petri dishes with LBK.<br />
#Spread 300 ul of the transformation onto first dish.<br />
#Added 900 ul of LB to 100 ul of the transformation and spread 300 ul of it onto second dish. <br />
#Incubated the plates at 37C for 15 hrs 45 min.<br />
</p><br />
<br />
==PCR==<br />
<p><br />
PCR to confirm Ag43-f4 primer.<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|1 ul<br />
|-<br />
|KOD-Plus-NEO(Taq polymerase)<br />
|1 ul<br />
|-<br />
|dNTP<br />
|5 ul<br />
|-<br />
|MgSO4<br />
|3 ul<br />
|-<br />
|KOD-Plus-NEO Buffer<br />
|5 ul<br />
|-<br />
|Ag43-f4 primer<br />
|1 ul<br />
|-<br />
|PS-R primer<br />
|1 ul<br />
|-<br />
|DW<br />
|33 ul<br />
|-<br />
|Total<br />
|50 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|94<br />
|120<br />
|-<br />
|2<br />
|98<br />
|10<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|68<br />
|60<br />
|-<br />
|5<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 45<br />
<br />
<br />
<br />
[[image:HokkaidoU2012 120803 Ag43 PCR(Forward=ag43-f4 Reverse=PS-R).jpg|thumb|PCR result]]<br />
<br />
From this result, we could see that the ag43-f4 primer had worked and DNA of last 500~600 bp of ag43 to BioBrick suffix were increased.<br />
</p><br />
<br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 4th==<br />
<div><br />
==Colony PCR==<br />
<p><br />
Colony PCR to confirm that whether the Ag43-dT was successfully ligated with pT7-RBS on pSB1K3 or not. <br />
<br />
<br />
{|class="hokkaidou-table-pcr-reagent"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|Kapa-Taq(Taq polymerase)<br />
|5 ul<br />
|-<br />
|Forward Primer(Ag43-f4 primer)<br />
|0.5 ul<br />
|-<br />
|Reverse Primer(PS-R primer)<br />
|0.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|95<br />
|120<br />
|-<br />
|2<br />
|95<br />
|30<br />
|-<br />
|3<br />
|58<br />
|30<br />
|-<br />
|4<br />
|72<br />
|60<br />
|-<br />
|5<br />
|72<br />
|60<br />
|-<br />
|6<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:2~4 x 35<br />
<br />
We used N1 (DW only) and N2 (Ag43-dT on pSB1AK3) as controls.<br />
Desired product is about 500~600bp.<br />
<br />
[[image:HokkaidoU2012 120804 pt7-rbs-ag43-dt coloP.jpg|thumb|Colony PCR result]]<br />
<br />
We thought that colonies No. 5 and 9 were exactly transformed into E. coli. and colony of No. 10 had high concentration band.<br />
Next step, we resuspended these three colonies and incubated. <br />
</p><br />
<br />
==Liquid culture==<br />
<p><br />
Liquid culture of colonies passed the colony PCR test.<br />
#Added 2 ml of LBK into culture tubes.<br />
#Resuspended 3 colonies.<br />
#Incubated the tubes at 37C for 13 hrs. <br />
</p><br />
<br />
</div></div><br />
<br />
<br />
<div class="hokkaidou-notebook-daily"><br />
==August 5th==<br />
<div><br />
==Plasmid extraction==<br />
<p><br />
Plasmid extraction of incubated medium from yesterday. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.<br />
<br />
[[image:HokkaidoU2012 120805 pt7-rbs-ag43-dt.jpg|thumb|Plasmid extraction results]]<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digested pT7-RBS on pSB1K3 and pT7-RBS (once digestioned with SpeI) with speI.<br />
<br />
<br />
pT7-RBS on pSB1K3<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|8 ul<br />
|-<br />
|SpeI<br />
|2 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|8 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
pT7-RBS (once digestioned with SpeI)<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|1 ul<br />
|-<br />
|DW<br />
|4 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|4 ul<br />
|-<br />
|SpeI<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|13 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 pT7-RBS digestion SpeI (no.jpg|thumb|digestion result]]<br />
From this result, the bottom band was digested incorrectly and middle band was digested correctly, we thought. Was the above band something contaminated in the DNA solution?<br />
<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction of middle band. We thought this band would be the exactly digested DNA fragment. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
==Sequencing==<br />
<p><br />
Sequencing to confirm what kind of DNA we made.<br />
<br />
<br />
{|<br />
|DNA<br />
|primer<br />
|-<br />
|Ag43 plasmid extraction product<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|K542009<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|-<br />
|pT7-RBS on pSB1C3<br />
|100bp-up forward primer<br />
|-<br />
|Ag43-dT on pSB1AK3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|Ag43-dT on pSB1T3<br />
|100bp-up forward primer,<br />
|ag43-f1,<br />
|ag43-f2,<br />
|ag43-f3,<br />
|ag43-f4<br />
|-<br />
|pT7-RBS on pSB1K3<br />
|100bp-up forward primer<br />
|}<br />
<br />
<br />
Sequencing PCR<br />
{|class="hokkaidou-table-sequencing-pcr-reagent"<br />
|-<br />
|template DNA<br />
|1 ul<br />
|-<br />
|Ready Reaction Premix<br />
|1 ul<br />
|-<br />
|5x Sequencing Buffer<br />
|1.5 ul<br />
|-<br />
|H2O<br />
|5 ul<br />
|-<br />
|Primer(1 pmol/ul)<br />
|1.5 ul<br />
|-<br />
|Total<br />
|10 ul<br />
|}<br />
<br />
<br />
{|class="hokkaidou-table-pcr-time"<br />
|-<br />
|Number<br />
|Degree<br />
|Second<br />
|-<br />
|1<br />
|96<br />
|10<br />
|-<br />
|2<br />
|50<br />
|5<br />
|-<br />
|3<br />
|60<br />
|240<br />
|-<br />
|4<br />
|4<br />
|HOLD<br />
|}<br />
Cycle:1~3 x 25<br />
<br />
<br />
To extract the PCR product, we did ethanol precipitation.<br />
<br><br />
Ethanol precipitation for sequencing.<br />
#Added 10 ul of H2O, 2 ul of NaoAc, 1 ul of glycogen and 50 ul of 100% ethanol.<br />
#Centrifuged at 15000 rpm, 15 min at 26C.<br />
#Removed supernatant and added 100ul of 70% ethanol.<br />
#Centrifuged at 15000 rpm, 5 min at 26C.<br />
#Removed supernatant and dried out at room temperature after that added 10 ul of Hi-Di. <br />
<br />
<br />
Then ran a sequencing machine.(ByoDye Terminator v3.1 Cycle Sequencing Kit)<br><br />
We could not get the sequencing data.<br />
</p><br />
<br />
==Digestion==<br />
<p><br />
Digesting of Ag43-dT (digested by SpeI and XbaI) by HindIII.<br />
<br />
{|class="hokkaidou-table-digestion"<br />
|-<br />
|DNA solution<br />
|10 ul<br />
|-<br />
|HindIII<br />
|1 ul<br />
|-<br />
|10xM buffer<br />
|2 ul<br />
|-<br />
|DW<br />
|7 ul<br />
|-<br />
|Total<br />
|20 ul<br />
|}<br />
<br />
<br />
[[image:HokkaidoU2012 120805 Ag43-dT on pSB1AK3(d+ with XbaI &amp; SpeI) digestion with HindIII.jpg|thumb|digestion result]]<br />
<br />
From this result, we confirmed that the pSB1AK3 was successfully digested by HindIII.<br />
</p><br />
<br />
==Gel extraction==<br />
<p><br />
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution. <br />
</p><br />
</div></div><br />
<!-- DO NOT EDIT UNDER THIS LINE @iTakeshi --><br />
</div><br />
<br style="line-height: 0; clear: both;" /><br />
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