http://2009.igem.org/wiki/index.php?title=Special:Contributions/TAI&feed=atom&limit=50&target=TAI&year=&month=2009.igem.org - User contributions [en]2024-03-28T08:37:27ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:TzuChiU_Formosa/ProtocolTeam:TzuChiU Formosa/Protocol2009-10-21T15:47:14Z<p>TAI: </p>
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==Protocol==<br />
<br />
https://static.igem.org/mediawiki/2009/e/ea/Project.jpg<br />
<br />
A. Culture CP919<br />
<br />
B. Put Aqueorin-GFP into plasmid .<br />
<br />
C. Transform the cp919 into the Midnight Apollo!<br />
<br />
D. Sense the light.<br />
<br />
E. In the dark, the Midnight Apollo! is activated and emit the light.<br />
<br />
<br />
====Competent Cell (CP919-Cph8)====<br />
#Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).<br />
#Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.<br />
#Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.<br />
#Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)<br />
#Spin down the bacteria at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 10ml FSB.<br />
#Keep the cells on ice for 3~4 hours.<br />
#Spin down, at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 5ml FSB.<br />
#Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf tubes. Freeze these tubes in liquid nitrogen, then transfer them to -80℃ freezer.<br />
<br />
<br />
<br />
====Tansformation Protocol ====<br />
<br />
#Take competent cell in an eppendorf tube from -80℃ freezer put in ice.<br />
#Add 2ul plasmid to competent cell and place in ice for 5 minutes.<br />
#Put the transformed cells into 42℃ water bath for 60 seconds.<br />
#Then place the cells in ice for 2 minutes.<br />
#Add 1ml LB to the cells and mixed.<br />
#Put the eppendorf tube in 37℃ water bath and incubate for an hour.<br />
#Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.<br />
#While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.<br />
#Incubate at 37℃ incubater for 16~18 hours.<br />
<br />
<br />
<br />
==== PCR ====<br />
<br />
<br />
1. Dissolve the primers in water to have the concentration of 10nM.<br />
<br />
<br />
2. PCR reaction mixer:<br />
<br />
Template DNA(10ng/μl) 5<br />
10× PCR buffer 2<br />
10× dNTP(2mM) 2<br />
forward primer(10μM) 0.5<br />
reverse primer(10μM) 0.5<br />
Pfu DNA polymerase(2Kb) 0.1<br />
PCR water 9.9<br />
_______________________________________<br />
Total 20 μl<br />
<br />
<br />
<br />
3. Put the reaction mixer in a PCR tube.<br />
<br />
4.The PCR program is as follow : <br />
:'''4.1'''<br />
:94℃ 30 seconds<br />
:60℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 9 times<br />
<br />
<br />
:'''4.2'''<br />
:94℃ 30 seconds<br />
:55℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 34 times<br />
<br />
Extend PCR product at 72℃ for 10 minutes <br />
<br />
<br />
5. The PCR product was examed by electrophoresis in 1% agarose.<br />
<br />
<br />
<br />
<br />
====T-A cloning====<br />
<br />
<br />
<br />
<br />
<br />
Take an eppendorf tube and add following component one by one and mix well,incubated at 4℃ overnight.<br />
<br />
<br />
2x ligase buffer 7.5μl<br />
Insert(Aeq.-GFP) 5.5μl<br />
Vector(pGEM-T-easy) 1μl<br />
T4 DNA exp 3/12 1μl<br />
_________________________________<br />
total 15μl<br />
<br />
<br />
====Cloning PCR(To verify the presence of our gene of interest)====<br />
<br />
<br />
<br />
<br />
<br />
#Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.<br />
#Discard the supernatant<br />
#Add 500μl ddH20, Vortex<br />
#Boil for 20 min.<br />
#Take 5μl of above bacterial lysate, and add 10x dNTP 2μl, 10x Buffer 2μl, forward primer(10μM)0.5μl reverse primer(10μM)0.5μl, Taq DNA polymerase 0.1μl, ddH20<br />
#Use the PCR to amplify our product:PCR program<br />
<br />
95℃ 4 min <br />
94℃ 30seconds <br />
55℃ 40seconds <br />
72℃ 2 min<br />
Cycle 34 times<br />
25℃ 2 min<br />
7. The PCR product was examed by electrophoresis in 1% agarose.<br />
<br />
<br />
====Plasmid extraction(Homemade)====<br />
<br />
<br />
<br />
<br />
<br />
#Transfer 1.5ml of bacterial culture to each well(24 wells plate)<br />
#pellet cells by centrifuging at 33000 rpm for 10min at 4℃<br />
#carefully remove the supernatant<br />
#add 100μl of Solution 1 (*)to each well, and vortex<br />
#add 200μl of Solution 2 (*)to each well, and mix gently<br />
#add 150μl of Solution 3 (*)to each well<br />
#centrifuge 3000rpm at 4℃ for 10min<br />
#add 1 ml 100% EtOH to new well<br />
#Transfer the supernatant to the new well, containing 100% EtOH.<br />
#centrifuge 3300rpm at 4℃ for 30min.<br />
#carefully remove the supernatant.<br />
#add 75% EtOH to wash pellets, then remove the supernantant, then air dry.<br />
#add 40μl ddH2O to each well, to dissolve with plasmid DNA<br />
#store the plate in 4℃<br />
(*)Sol 1: 10m Miris/0.5mM HEDTA, PH 7.4 buffer.<br />
Sol 2: 0.2N NaOH/1% SDS<br />
Sol 3: 3M KOAC/HOAC<br />
<br />
====Digestion====<br />
<br />
<br />
<br />
<br />
Digestion mixture <br />
<br />
DNA 20μl<br />
10x buffer 3μl<br />
Enzyme 1μl<br />
RNase H2O 6μl<br />
__________________________<br />
Total 30μl<br />
3. The order for adding materials to wells is from plenty to less<br />
4. Place the plate in 37℃water bath overnight</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ProtocolTeam:TzuChiU Formosa/Protocol2009-10-21T15:39:29Z<p>TAI: </p>
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<br />
==Protocol==<br />
<br />
https://static.igem.org/mediawiki/2009/e/ea/Project.jpg<br />
<br />
A. Culture CP919<br />
<br />
B. Put Aqueorin-GFP into plasmid .<br />
<br />
C. Transform the cp919 into the Midnight Apollo!<br />
<br />
D. Sense the light.<br />
<br />
E. In the dark, the Midnight Apollo! is activated and emit the light.<br />
<br />
<br />
====Competent Cell (CP919-Cph8)====<br />
#Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).<br />
#Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.<br />
#Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.<br />
#Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)<br />
#Spin down the bacteria at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 10ml FSB.<br />
#Keep the cells on ice for 3~4 hours.<br />
#Spin down, at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 5ml FSB.<br />
#Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf tubes. Freeze these tubes in liquid nitrogen, then transfer them to -80℃ freezer.<br />
<br />
<br />
<br />
====Tansformation Protocol ====<br />
<br />
#Take competent cell in an eppendorf tube from -80℃ freezer put in ice.<br />
#Add 2ul plasmid to competent cell and place in ice for 5 minutes.<br />
#Put the transformed cells into 42℃ water bath for 60 seconds.<br />
#Then place the cells in ice for 2 minutes.<br />
#Add 1ml LB to the cells and mixed.<br />
#Put the eppendorf tube in 37℃ water bath and incubate for an hour.<br />
#Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.<br />
#While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.<br />
#Incubate at 37℃ incubater for 16~18 hours.<br />
<br />
<br />
<br />
==== PCR ====<br />
<br />
<br />
1. Dissolve the primers in water to have the concentration of 10nM.<br />
<br />
<br />
2. PCR reaction mixer:<br />
<br />
Template DNA(10ng/μl) 5<br />
10× PCR buffer 2<br />
10× dNTP(2mM) 2<br />
forward primer(10μM) 0.5<br />
reverse primer(10μM) 0.5<br />
Pfu DNA polymerase(2Kb) 0.1<br />
PCR water 9.9<br />
_______________________________________<br />
Total 20 μl<br />
<br />
<br />
<br />
3. Put the reaction mixer in a PCR tube.<br />
<br />
4.The PCR program is as follow : <br />
:'''4.1'''<br />
:94℃ 30 seconds<br />
:60℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 9 times<br />
<br />
<br />
:'''4.2'''<br />
:94℃ 30 seconds<br />
:55℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 34 times<br />
<br />
Extend PCR product at 72℃ for 10 minutes <br />
<br />
<br />
5. The PCR product was examed by electrophoresis in 1% agarose.<br />
<br />
<br />
<br />
<br />
====T-A cloning====<br />
<br />
<br />
<br />
<br />
<br />
Take an eppendorf tube and add following component one by one and mix well,incubated at 4℃ overnight.<br />
<br />
<br />
2x ligase buffer 7.5μl<br />
Insert(Aeq.-GFP) 5.5μl<br />
Vector(pGEM-T-easy) 1μl<br />
T4 DNA exp 3/12 1μl<br />
_________________________________<br />
total 15μl<br />
<br />
<br />
====Cloning PCR(To verify the presence of our gene of interest)====<br />
<br />
<br />
<br />
<br />
<br />
#Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.<br />
#Discard the supernatant<br />
#Add 500μl ddH20, Vortex<br />
#Boil for 20 min.<br />
#Take 5μl of above bacterial lysate, and add 10x dNTP 2μl, 10x Buffer 2μl, forward primer(10μM)0.5μl reverse primer(10μM)0.5μl, Taq DNA polymerase 0.1μl, ddH20<br />
#Use the PCR to amplify our product:PCR program<br />
<br />
95℃ 4 min <br />
94℃ 30seconds <br />
55℃ 40seconds <br />
72℃ 2 min<br />
Cycle 34 times<br />
25℃ 2 min<br />
7. The PCR product was examed by electrophoresis in 1% agarose.<br />
<br />
<br />
====Plasmid extraction(Homemade)====<br />
<br />
<br />
<br />
<br />
<br />
1. Transfer 1.5ml of bacterial culture to each well(24 wells plate)<br />
2. pellet cells by centrifuging at 33000 rpm for 10min at 4℃<br />
3. carefully remove the supernatant<br />
4. add 100μl of Solution 1 (*)to each well, and vortex<br />
5. add 200μl of Solution 2 (*)to each well, and mix gently<br />
6. add 150μl of Solution 3 (*)to each well<br />
7. centrifuge 3000rpm at 4℃ for 10min<br />
8. add 1 ml 100% EtOH to new well<br />
9. Transfer the supernatant to the new well, containing 100% EtOH.<br />
10. centrifuge 3300rpm at 4℃ for 30min.<br />
11. carefully remove the supernatant.<br />
12. add 75% EtOH to wash pellets, then remove the supernantant, then air dry.<br />
13. add 40μl ddH2O to each well, to dissolve with plasmid DNA<br />
14. store the plate in 4℃<br />
(*)Sol 1: 10m Miris/0.5mM HEDTA, PH 7.4 buffer.<br />
Sol 2: 0.2N NaOH/1% SDS<br />
Sol 3: 3M KOAC/HOAC<br />
<br />
====Digestion=====<br />
<br />
<br />
<br />
<br />
Digestion mixture <br />
<br />
DNA 20μl<br />
10x buffer 3μl<br />
Enzyme 1μl<br />
RNase H2O 6μl<br />
__________________________<br />
Total 30μl<br />
3. The order for adding materials to wells is from plenty to less<br />
4. Place the plate in 37℃water bath overnight</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ResultTeam:TzuChiU Formosa/Result2009-10-21T15:33:57Z<p>TAI: </p>
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<br />
==Result==<br />
<gallery><br />
Image:Result_f1a.jpg|a<br />
Image:Result_f1b.jpg|b<br />
Image:Result_f1c.jpg|c<br />
</gallery><br />
'''Figure.1(a.)pSB1A3 cloning PCR gel image. start with left to right part:(1)100 bps marker,(2)ompC promoter,(3)ompC promoter,(4)Negative control。(b.)the three red circles are single colony that have been transformed OmpC promoter containing plasmid。(c.)OmpC plasmid gel image。start with left to right part:(1)100 bps marker,(2)pSB1A3 plasmid,(3)pSB1A3 plasmid.'''<br />
<br />
<br />
Our PCR show that pSBA1B3 contained OmpC promoter, and the size was about 108 base pairs. From Fig.1-a Show that band was placed at 108 base pairs, which confirmed that pSB1A3 contained OmpC promoter. From Fig1-b we can see the growing of single colony. The pSB1A3 plasmid size was about 2.75 Kb, Fig1-c shows plasmid was at 2.75 Kb, so it was confirmed that pSB1A3 had been successfully transformed into DH5-α competent cell. <br />
<br />
<gallery><br />
Image:Result_f2a.jpg|a<br />
Image:Result_f2b.jpg|b<br />
Image:Result_f2c.jpg|c<br />
</gallery><br />
'''Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(1)100 bps marker,(2)Aequorin-GFP.(b.)After TA cloning transformation into DH5-αcompetent cell,spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image.Start with left to right:(1)100 bps marker,the other(2)Aequorin-GFP.<br />
'''<br />
<br />
<br />
Because of Aequorin-GFP didn't know the purify and concentration;Through PCR, we run the purify step to increasing the purify. The Aequorin-GFP size was about 2000 bps;Fig.2-a show that template was placed at 2000 bps, so it confirm that suecssfully purify the Aequorin-GFP. And took the successful purify Aequorin-GFP to run the TA cloning, let Aequorin-GFP could ligate to pGEM-T-easy plasmid. After transformation into DH5-α competent cell, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we picked from the two closing size single colony after put at the 37℃ and amplify, then run cloning PCR show that Aequorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp,so it confirm Aequorin-GFP had been successfully ligate to Pgem-T-easy plasmid.<br />
<br />
<gallery><br />
Image:Result_f3a.jpg|a<br />
Image:Result_f3b.JPG|b<br />
</gallery><br />
'''Figure.3(a.)PSB1A3 digestion gel image.Start with left to right:(1)100b.p.marker, (2)None cut PSB1A3, (2)None cut PSB1A3, (3)cut PSB1A3.(b.) (1) 100bps marker (2) Dephosphorelation of Pst1 cutting site in pSB1A3.<br />
<br />
'''<br />
<br />
<br />
When we confirm the PSB1A3 contain OmpC promoter. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.<br />
<br />
In order to improve the successful rate of putting insert into the vector we treated, Pst1 digested pSB1A3 with calf Intestinal Alkaline Phosphatase (CIP) to remove the phosphate group thus reducing the chance of self ligation</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ResultTeam:TzuChiU Formosa/Result2009-10-21T15:24:56Z<p>TAI: </p>
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<br />
==Result==<br />
<gallery><br />
Image:Result_f1a.jpg|a<br />
Image:Result_f1b.jpg|b<br />
Image:Result_f1c.jpg|c<br />
</gallery><br />
'''Figure.1(a.)pSB1A3 cloning PCR gel image. start with left to right part:(m.)100 bps marker,(C)ompC promoter,(C)ompC promoter,(N)Negative control。(b.)the three red circles are single colony that have been transformed OmpC promoter containing plasmid。(c.)OmpC plasmid gel image。start with left to right part:(M)100 bps marker,(PS)pSB1A3 plasmid,(PS)pSB1A3 plasmid.'''<br />
<br />
<br />
Our PCR show that pSBA1B3 contained OmpC promoter, and the size was about 108 base pairs. From Fig.1-a Show that band was placed at 108 base pairs, which confirmed that pSB1A3 contained OmpC promoter. From Fig1-b we can see the growing of single colony. The pSB1A3 plasmid size was about 2.75 Kb, Fig1-c shows plasmid was at 2.75 Kb, so it was confirmed that pSB1A3 had been successfully transformed into DH5-α competent cell. <br />
<br />
<gallery><br />
Image:Result_f2a.jpg|a<br />
Image:Result_f2b.jpg|b<br />
Image:Result_f2c.jpg|c<br />
</gallery><br />
'''Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(M)100 bps marker,(AG)Aequorin-GFP.(b.)After TA cloning transformation into DH5-αcompetent cell,spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image.Start with left to right:(M)100 bps marker,the other(A-G)Aequorin-GFP.<br />
'''<br />
<br />
<br />
Because of Aequorin-GFP didn't know the purify and concentration;Through PCR, we run the purify step to increasing the purify. The Aequorin-GFP size was about 2000 bps;Fig.2-a show that template was placed at 2000 bps, so it confirm that suecssfully purify the Aequorin-GFP. And took the successful purify Aequorin-GFP to run the TA cloning, let Aequorin-GFP could ligate to pGEM-T-easy plasmid. After transformation into DH5-α competent cell, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we picked from the two closing size single colony after put at the 37℃ and amplify, then run cloning PCR show that Aequorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp,so it confirm Aequorin-GFP had been successfully ligate to Pgem-T-easy plasmid.<br />
<br />
<gallery><br />
Image:Result_f3a.jpg|a<br />
Image:Result_f3b.JPG|b<br />
</gallery><br />
'''Figure.3(a.)PSB1A3 digestion gel image.Start with left to right:(M)100b.p.marker, (1)None cut PSB1A3, (2)None cut PSB1A3, (3)cut PSB1A3.'''<br />
<br />
<br />
When we confirm the PSB1A3 contain OmpC promoter. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.<br />
<br />
In order to improve the successful rate of putting insert into the vector we treated, Pst1 digested pSB1A3 with calf Intestinal Alkaline Phosphatase (CIP) to remove the phosphate group thus reducing the chance of self ligation</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ResultTeam:TzuChiU Formosa/Result2009-10-21T13:50:32Z<p>TAI: /* Result */</p>
<hr />
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<body><br />
<p><br />
<object type="application/x-shockwave-flash" height="240" width="960" data="https://static.igem.org/mediawiki/2009/a/a2/IGEM_s.swf"><br />
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<br />
==Result==<br />
<gallery><br />
Image:Result_f1a.jpg|a<br />
Image:Result_f1b.jpg|b<br />
Image:Result_f1c.jpg|c<br />
</gallery><br />
'''Figure.1(a.)SB1A3 cloning PCR gel image. start with left to right part:(1)100b.p. marker,(2)PSB1A3,(3)PSB1A3,(4)Negative control。(b.)the plate is PSB1A3 transformation into DH5α’s single colony。(c.)ompC plasmid gel image。start with left to right part:(1)100b.p. marker,(2)ompC promoter,(3)ompC promoter。'''<br />
<br />
<br />
Our PCR show that PSBA1B3 contained OmpC promoter, and the size was about 108 base pairs. From Fig.1-a Show that template was placed at 108 base pairs, which confirmed that PSB1A3 contained OmpC promoter. After transforming PSB1A3 into DH5a , culturing and spirting; From Fig1-b we can see the growing of single colony. Picking two similar size of single colonies, after being cultured in 37℃ and being amplified, draw out PSB1A3 plasmid and run gel electrophoresis to check if the transformation was succeed or not. The PSB1A3 size was about 2.75kb; Fig1-c shows plasmid was at 2.75kb, so it was confirmed that PSB1A3 had been successfully transformed into DH5a. <br />
<br />
<br />
<gallery><br />
Image:Result_f2a.jpg|a<br />
Image:Result_f2b.jpg|b<br />
Image:Result_f2c.jpg|c<br />
</gallery><br />
'''Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(1)100b.p. marker,(2)Aequorin-GFP。(b.)After TA cloning transformation into DH5α,spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image。Start with left to right:(1)100b.p.marker,the other(A-G)Aequorin-GFP。<br />
'''<br />
<br />
<br />
Because of Aequorin-GFP didn't know the purify and concentration;Through PCR, we run the purify step to increasing the purify. The Aequorin-GFP size was about 2000bp;Fig.2-a show that template was placed at 2000 bp,so it confirm that suecssfully purify the Aequorin-GFP. And took the successful purify Aequorin-GFP to run the TA cloning, let Aequorin-GFP could ligate to Pgem-T-easy plasmid. After transformation into DH5a, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we picked from the two closing size single colony after put at the 37℃ and amplify, then run cloning PCR show that Aequorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp,so it confirm Aequorin-GFP had been successfully ligate to Pgem-T-easy plasmid。<br />
<br />
<br />
<gallery><br />
Image:Result_f3a.jpg|a<br />
Image:Result_f3b.JPG|b<br />
</gallery><br />
'''Figure.3(a.)PSB1A3 digestion gel image.Start with left to right:(M)100b.p.marker, (1)None cut PSB1A3, (2)None cut PSB1A3, (3)cut PSB1A3.'''<br />
<br />
<br />
When we confirm the PSB1A3 contain OmpC promoter. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.<br />
<br />
In order to improve the successful rate of putting insert into the vector we treated, Pst1 digested pSB1A3 with calf Intestinal Alkaline Phosphatase (CIP) to remove the phosphate group thus reducing the chance of self ligation</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ResultTeam:TzuChiU Formosa/Result2009-10-21T13:48:34Z<p>TAI: </p>
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<br />
==Result==<br />
<gallery><br />
Image:Result_f1a.jpg|a<br />
Image:Result_f1b.jpg|b<br />
Image:Result_f1c.jpg|c<br />
</gallery><br />
'''Figure.1(a.)SB1A3 cloning PCR gel image. start with left to right part:(1)100b.p. marker,(2)PSB1A3,(3)PSB1A3,(4)Negative control。(b.)the plate is PSB1A3 transformation into DH5α’s single colony。(c.)ompC plasmid gel image。start with left to right part:(1)100b.p. marker,(2)ompC promoter,(3)ompC promoter。'''<br />
<br />
<br />
Our PCR show that PSBA1B3 contained OmpC promoter, and the size was about 108 base pairs. From Fig.1-a Show that template was placed at 108 base pairs, which confirmed that PSB1A3 contained OmpC promoter. After transforming PSB1A3 into DH5a , culturing and spirting; From Fig1-b we can see the growing of single colony. Picking two similar size of single colonies, after being cultured in 37℃ and being amplified, draw out PSB1A3 plasmid and run gel electrophoresis to check if the transformation was succeed or not. The PSB1A3 size was about 2.75kb; Fig1-c shows plasmid was at 2.75kb, so it was confirmed that PSB1A3 had been successfully transformed into DH5a. <br />
<br />
<br />
<gallery><br />
Image:Result_f2a.jpg|a<br />
Image:Result_f2b.jpg|b<br />
Image:Result_f2c.jpg|c<br />
</gallery><br />
'''Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(1)100b.p. marker,(2)Aequorin-GFP。(b.)After TA cloning transformation into DH5α,spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image。Start with left to right:(1)100b.p.marker,the other(A-G)Aequorin-GFP。<br />
'''<br />
<br />
<br />
Because of Aequorin-GFP didn't know the purify and concentration;Through PCR, we run the purify step to increasing the purify. The Aequorin-GFP size was about 2000bp;Fig.2-a show that template was placed at 2000 bp,so it confirm that suecssfully purify the Aequorin-GFP. And took the successful purify Aequorin-GFP to run the TA cloning, let Aequorin-GFP could ligate to Pgem-T-easy plasmid. After transformation into DH5a, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we picked from the two closing size single colony after put at the 37℃ and amplify, then run cloning PCR show that Aequorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp,so it confirm Aequorin-GFP had been successfully ligate to Pgem-T-easy plasmid。<br />
<br />
<br />
<gallery><br />
Image:Result_f3a.jpg|a<br />
Image:Result_f3b.JPG|b<br />
</gallery><br />
'''Figure.3(a.)PSB1A3 digestion gel image.Start with left to right:(M)100b.p.marker, (1)None cut PSB1A3, (2)None cut PSB1A3, (3)cut PSB1A3.'''<br />
<br />
<br />
When we confirm the PSB1A3 contain OmpC promoter. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.</div>TAIhttp://2009.igem.org/File:Result_f3b.JPGFile:Result f3b.JPG2009-10-21T13:47:18Z<p>TAI: </p>
<hr />
<div></div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/DiscussionTeam:TzuChiU Formosa/Discussion2009-10-21T13:38:28Z<p>TAI: /* Discussion */</p>
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<br />
==Discussion==<br />
<br />
<br />
<br />
<br />
Aequorin-GFP coding Sequence containing plasmid was kindly provide by French scientist() We use PCR to Pst1 site at both ends of Aequorin-GFP coding Sequence. Then we insert Aequorin-GFP lst into TA vector to have easier cloning step3 to have it inserted in out ompC promoter plasmid. <br />
Because cloning into TA vector will allow us to ohtc equate am of Aequorin-GFP into ompC promoter plasmid. We have to determine the orientation of the Aequorin-GFP insert and choice the rights 5’to3’ orientation.<br />
<br />
Dues to late start of our project will we still in our project in trying to insert A-G into PSB1A3 we hope we will successfully construct the plasmid we need and transform into CP919. Finally we will determine the optimal Ca2+ concentrations that can have maximal illuminating of A-G in bacteria to create aim biolight .</div>TAIhttp://2009.igem.org/File:Result_f3a.jpgFile:Result f3a.jpg2009-10-21T13:30:36Z<p>TAI: uploaded a new version of "Image:Result f3a.jpg"</p>
<hr />
<div></div>TAIhttp://2009.igem.org/File:Result_f2c.jpgFile:Result f2c.jpg2009-10-21T13:28:53Z<p>TAI: uploaded a new version of "Image:Result f2c.jpg"</p>
<hr />
<div></div>TAIhttp://2009.igem.org/File:Result_f2a.jpgFile:Result f2a.jpg2009-10-21T13:27:29Z<p>TAI: uploaded a new version of "Image:Result f2a.jpg"</p>
<hr />
<div></div>TAIhttp://2009.igem.org/File:Result_f1c.jpgFile:Result f1c.jpg2009-10-21T13:25:52Z<p>TAI: uploaded a new version of "Image:Result f1c.jpg"</p>
<hr />
<div></div>TAIhttp://2009.igem.org/File:Result_f1a.jpgFile:Result f1a.jpg2009-10-21T13:21:16Z<p>TAI: uploaded a new version of "Image:Result f1a.jpg"</p>
<hr />
<div></div>TAIhttp://2009.igem.org/File:Result_f1a.jpgFile:Result f1a.jpg2009-10-21T13:16:03Z<p>TAI: uploaded a new version of "Image:Result f1a.jpg"</p>
<hr />
<div></div>TAIhttp://2009.igem.org/File:Result_f1a.jpgFile:Result f1a.jpg2009-10-21T13:12:30Z<p>TAI: uploaded a new version of "Image:Result f1a.jpg"</p>
<hr />
<div></div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ResultTeam:TzuChiU Formosa/Result2009-10-20T14:10:17Z<p>TAI: </p>
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<br />
==Result==<br />
<gallery><br />
Image:Result_f1a.jpg|a<br />
Image:Result_f1b.jpg|b<br />
Image:Result_f1c.jpg|c<br />
</gallery><br />
'''Figure.1(a.)SB1A3 cloning PCR gel image. start with left to right part:(b.)100b.p. marker,(PS)PSB1A3,(PS)PSB1A3,(N)Negative control。(b.)the plate is PSB1A3 transformation into DH5α’s single colony。(c.)ompC plasmid gel image。start with left to right part:(M)100b.p. marker,(C)ompC promoter,(C)ompC promoter。'''<br />
<br />
<br />
Our PCR show that PSBA1B3 contained OmpC promoter, and the size was about 108 base pairs. From Fig.1-a Show that template was placed at 108 base pairs, which confirmed that PSB1A3 contained OmpC promoter. After transforming PSB1A3 into DH5a , culturing and spirting; From Fig1-b we can see the growing of single colony. Picking two similar size of single colonies, after being cultured in 37℃ and being amplified, draw out PSB1A3 plasmid and run gel electrophoresis to check if the transformation was succeed or not. The PSB1A3 size was about 2.75kb; Fig1-c shows plasmid was at 2.75kb, so it was confirmed that PSB1A3 had been successfully transformed into DH5a. <br />
<br />
<br />
<gallery><br />
Image:Result_f2a.jpg|a<br />
Image:Result_f2b.jpg|b<br />
Image:Result_f2c.jpg|c<br />
</gallery><br />
'''Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(M)100b.p. marker,(AG)Aequorin-GFP。(b.)After TA cloning transformation into DH5α,spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image。Start with left to right:(M)100b.p.marker,the other(A-G)Aequorin-GFP。<br />
'''<br />
<br />
<br />
Because of Aequorin-GFP didn't know the purify and concentration;Through PCR, we run the purify step to increasing the purify. The Aequorin-GFP size was about 2000bp;Fig.2-a show that template was placed at 2000 bp,so it confirm that suecssfully purify the Aequorin-GFP. And took the successful purify Aequorin-GFP to run the TA cloning, let Aequorin-GFP could ligate to Pgem-T-easy plasmid. After transformation into DH5a, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we picked from the two closing size single colony after put at the 37℃ and amplify, then run cloning PCR show that Aequorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp,so it confirm Aequorin-GFP had been successfully ligate to Pgem-T-easy plasmid。<br />
<br />
<br />
<gallery><br />
Image:Result_f3a.jpg|a<br />
</gallery><br />
'''Figure.3(a.)PSB1A3 digestion gel image.Start with left to right:(M)100b.p.marker, (None)None cut PSB1A3, (None)None cut PSB1A3, (cut)cut PSB1A3.'''<br />
<br />
<br />
When we confirm the PSB1A3 contain OmpC promoter. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ResultTeam:TzuChiU Formosa/Result2009-10-20T13:53:24Z<p>TAI: </p>
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<br />
==Result==<br />
<gallery><br />
Image:Result_f1a.jpg|a<br />
Image:Result_f1b.jpg|b<br />
Image:Result_f1c.jpg|c<br />
</gallery><br />
'''Figure.1(a.)SB1A3 cloning PCR gel image. start with left to right part:(b.)100b.p. marker,(PS)PSB1A3,(PS)PSB1A3,(N)Negative control。(b.)the plate is PSB1A3 transformation into DH5α’s single colony。(c.)ompC plasmid gel image。start with left to right part:(M)100b.p. marker,(C)ompC promoter,(C)ompC promoter。'''<br />
<br />
<br />
According to cloning PCR experiment we could make sure that PSB1A3 contained OmpC promoter, and the size of OmpC promoter was about 108 base pair. From Fig.1-a we could know that template was placed at 108 bp, which confirmed that PSB1A3 really contained OmpC promoter. After transforming PSB1A3 into DH5a , culturing and spirting; From Fig1-b we can see the growing of single colony. Picking two similar size of single colonies, after being cultured in 37℃ and being amplified, draw out PSB1A3 plasmid and run gel electrophoresis to check if the transformation was succeed or not. The PSB1A3 size was about 2.75kb; Fig1-c shows plasmid was at 2.75kb, so it was confirmed that PSB1A3 had been successfully transformed into DH5a. <br />
<br />
<br />
<gallery><br />
Image:Result_f2a.jpg|a<br />
Image:Result_f2b.jpg|b<br />
Image:Result_f2c.jpg|c<br />
</gallery><br />
'''Figure.2(a.)Aequorin-GFP sequence after purify gel image。Left:(M)100b.p. marker,(AG)Aequorin-GFP。(b.)After TA cloning transformation into DH5α,spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image。Start with left to right:(M)100b.p.marker,the other(A-G)Aequorin-GFP。<br />
'''<br />
<br />
<br />
Because of Aequorin-GFP didn't know the purify and concentration;Through PCR, we run the purify step to increasing the purify. The Aequorin-GFP size was about 2000bp;Fig.2-a show that template was placed at 2000 bp,so it confirm that suecssfully purify the Aequorin-GFP. And took the successful purify Aequorin-GFP to run the TA cloning, let Aequorin-GFP could ligate to Pgem-T-easy plasmid. After transformation into DH5a, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we picked from the two closing size single colony after put at the 37℃ and amplify, then run cloning PCR show that Aequorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp,so it confirm Aequorin-GFP had been successfully ligate to Pgem-T-easy plasmid。<br />
<br />
<br />
<gallery><br />
Image:Result_f3a.jpg|a<br />
</gallery><br />
'''Figure.3(a.)PSB1A3 digestion gel image.Start with left to right:(M)100b.p.marker, (None)None cut PSB1A3, (None)None cut PSB1A3, (cut)cut PSB1A3.'''<br />
<br />
<br />
When we confirm the PSB1A3 contain OmpC. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ProtocolTeam:TzuChiU Formosa/Protocol2009-10-20T13:48:34Z<p>TAI: </p>
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<br />
==Protocol==<br />
<br />
https://static.igem.org/mediawiki/2009/e/ea/Project.jpg<br />
<br />
====Tansformation Protocol ====<br />
<br />
#Take the cp919 competent cell in an eppendorf tube from -80℃ freezer put in ice.<br />
#Add 4ul plasmid to competent cell and place in ice for 30 minutes.<br />
#Put the transformed cells into 42℃ water bath for 90 seconds.<br />
#Then place the cells in ice for 2 minutes.<br />
#Add 1ml LB to the cells and mixed.<br />
#Put the eppendorf tube in 37℃ water bath and incubate for an hour.<br />
#Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.<br />
#While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.<br />
#Incubate at 37℃ incubater for 16~18 hours.<br />
<br />
====Competent Cell (CP919-Cph8)====<br />
#Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).<br />
#Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.<br />
#Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.<br />
#Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)<br />
#Spin down the bacteria at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 10ml FSB.<br />
#Keep the cells on ice for 3~4 hours.<br />
#Spin down, at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 5ml FSB.<br />
#Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf tubes. Freeze these tubes in liquid nitrogen, then transfer them to -80℃ freezer.<br />
<br />
<br />
==== PCR ====<br />
<br />
<br />
1. Dissolve the primers in water to have the concentration of 10nM.<br />
<br />
<br />
2. PCR reaction mixer:<br />
<br />
Template DNA(10ng/μl) 5<br />
10× PCR buffer 2<br />
10× dNTP(2mM) 2<br />
forward primer(10μM) 0.5<br />
reverse primer(10μM) 0.5<br />
Pfu DNA polymerase(2Kb) 0.1<br />
PCR water 9.9<br />
_______________________________________<br />
Total 20 μl<br />
<br />
<br />
<br />
3. Put the reaction mixer in a PCR tube.<br />
<br />
4.The PCR program is as follow : <br />
:'''4.1'''<br />
:94℃ 30 seconds<br />
:60℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 9 times<br />
<br />
<br />
:'''4.2'''<br />
:94℃ 30 seconds<br />
:55℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 34 times<br />
<br />
Extend PCR product at 72℃ for 10 minutes <br />
<br />
<br />
5. The PCR product was examed by electrophoresis in 1% agarose.<br />
<br />
<br />
<br />
<br />
====T-A cloning protocol====<br />
<br />
<br />
Take an eppendorf tube and add following component one by one and mix well,incubated at 4℃ overnight.<br />
<br />
<br />
2x ligase buffer 7.5μl<br />
Insert(Aeq.-GFP) 5.5μl<br />
Vector(pGEM-T-easy) 1μl<br />
T4 DNA exp 3/12 1μl<br />
_________________________________<br />
total 15μl<br />
<br />
<br />
====Cloning PCR(To verify the presence of our gene of interest)====<br />
#Take 10μl of bacterial culture, spin down at 14000 rpm for 10 min.<br />
#Discard the supernatant<br />
#Add 500μl ddH20, Vortex<br />
#Boil for 20 min.<br />
#Take 5μl of above bacterial lysate, and add 10x dNTP 2μl, 10x Buffer 2μl, forward primer(10μM)0.5μl reverse primer(10μM)0.5μl, Taq DNA polymerase 0.1μl, ddH20<br />
#Use the PCR to amplify our product:PCR program<br />
<br />
95℃ 4 min <br />
94℃ 30seconds <br />
55℃ 40seconds <br />
72℃ 2 min<br />
Cycle 34 times<br />
25℃ 2 min<br />
7. The PCR product was examed by electrophoresis in 1% agarose.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ProtocolTeam:TzuChiU Formosa/Protocol2009-10-20T13:25:24Z<p>TAI: </p>
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<br />
==Protocol==<br />
<br />
https://static.igem.org/mediawiki/2009/e/ea/Project.jpg<br />
<br />
====Tansformation Protocol ====<br />
<br />
#Take the cp919 competent cell in an eppendorf tube from -80℃ freezer put in ice.<br />
#Add 4ul plasmid to competent cell and place in ice for 30 minutes.<br />
#Put the transformed cells into 42℃ water bath for 90 seconds.<br />
#Then place the cells in ice for 2 minutes.<br />
#Add 1ml LB to the cells and mixed.<br />
#Put the eppendorf tube in 37℃ water bath and incubate for an hour.<br />
#Spin down at 7000 rpm for 5 minutes and remove most of the supernatant.<br />
#While the cells are incubated at 37℃ water bath spread 100ul Ampicilin(50mg/ml) on the plate. When the plate is dried, spread bacteria on the plate.<br />
#Incubate at 37℃ incubater for 16~18 hours.<br />
<br />
====Competent Cell (CP919-Cph8)====<br />
#Day1. Streak out the E.coli strain on an LB plate (with kanamycin)and incubate at 37℃ overnight (16-20 hours).<br />
#Day2. Select a single colony and inoculate 10 ml sterile LB and grow overnight (16-20 hours) in a 37℃ shaker incubator.<br />
#Day3. Add 2ml overnight culture to 250ml flask containing 100 ml LB.<br />
#Grow the cultures to OD600 = 0.2~0.4 (incubate with shaking for 75~90 min.)<br />
#Spin down the bacteria at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 10ml FSB.<br />
#Keep the cells on ice for 3~4 hours.<br />
#Spin down, at 4℃,3000 rpm for 10 min.<br />
#Discard the supernatant and mix the cell pellet with 5ml FSB.<br />
#Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf tubes. Freeze these tubes in liquid nitrogen, then transfer them to -80℃ freezer.<br />
<br />
<br />
====Real Time PCR====<br />
<br />
<br />
1. Take ice for preparing.<br />
<br />
2. dilution of concentration:<br />
<br />
:'''2.1'''<br />
:template DNA(Omp R):500 times diluted <br />
:499μl ddH2O + 1μl omp R=500μl<br />
<br />
:'''2.2'''<br />
:primer forward & primer reverse(p88):10 times diluted<br />
:10μl p88 primer forward+ 90μl ddH2O<br />
<br />
3. Materials:<br />
<br />
Template DNA(10ng/μl) 5<br />
10× PCR buffer 2<br />
10× dNTP(2mM) 2<br />
forward primer(10μM) 0.5<br />
reverse primer(10μM) 0.5<br />
Pfu DNA polymerase(2Kb) 0.1<br />
PCR water 9.9<br />
_______________________________________<br />
Total 20 μl<br />
<br />
4. Step:<br />
<br />
Meacure template DNA 2μl<br />
Measure 10× PCR buffer 2μl<br />
Measure 10× dNTP 2μl<br />
Measure forward primer 0.5μl<br />
Measure reverse primer 0.5μl<br />
Measure Pfu DNA polymerase 0.1μl<br />
Follow this step ,puttng the reagent in the eppendorf.<br />
<br />
5. Use the PCR to amplify our product:PCR program<br />
<br />
<br />
:'''5.1'''<br />
:94℃ 30 seconds<br />
:60℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 9 times<br />
<br />
<br />
:'''5.2'''<br />
:94℃ 30 seconds<br />
:55℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 34 times<br />
:72℃ 10 minutes<br />
<br />
<br />
6. The PCR production separate by 1% agar.<br />
<br />
7. After separate using the EtBr to dye agar and exposing by UV.<br />
<br />
<br />
====T-A cloning protocol====<br />
<br />
<br />
Take an eppendorf,and add these one by one,after mix well,overnight 4℃.<br />
<br />
<br />
2x ligase buffer 7.5μl<br />
Insert(Aeq.-GFP) 5.5μl<br />
Vector(pGEM-T-easy) 1μl<br />
T4 DNA exp 3/12 1μl<br />
_________________________________<br />
total 15μl<br />
<br />
<br />
====Cloning PCR====<br />
#Add 10μl cell, centrifuge 14000 rpm, 10 min<br />
#Discard the supernatant<br />
#Add 500μl ddH20, Votex<br />
#Boiling 20min, 100℃<br />
#Add 5μl DNA, 10x dNTP 2μl, 10x Buffer 2μl, forward primer(10μM)0.5μl reverse primer(10μM)0.5μl, Taq DNA polymerase 0.1μl, ddH20<br />
#Use the PCR to amplify our product:PCR program<br />
<br />
95℃ 4 min <br />
94℃ 30seconds <br />
55℃ 40seconds <br />
72℃ 2 min<br />
Cycle 34 times<br />
25℃ 2 min<br />
7. The PCR production separate by 1% agar. <br />
<br />
8. After separate using the EtBr to dye agar and exposing by UV.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ProtocolTeam:TzuChiU Formosa/Protocol2009-10-20T13:04:08Z<p>TAI: /* Tansformation Protocol Useing Heat Shook */</p>
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<br />
==Protocol==<br />
<br />
https://static.igem.org/mediawiki/2009/e/ea/Project.jpg<br />
<br />
====Tansformation Protocol ====<br />
<br />
#Take the cp919 competent cell eppendorf from -80℃ freezer.<br />
#Add 4ul plasmid to competent cell and place at ice for 30 minutes.<br />
#Put the eppendorf into 42℃ water bath for 90 seconds.<br />
#Place the eppendorf at ice for 2 minutes.<br />
#Add 1ml LBM to the eppendorf and well mixed.<br />
#Put the eppendorf into 37℃ water bath incubate for an hour.<br />
#Centrifuged at 7000 r.p.m for 5 minutes and remove some supernatant liquid.<br />
#Spread antibiotic which we need on plate. When the plate dry, spread appropriate the culture liquid on plate.<br />
#Incubate into 37℃ water bath for 16~18 hours.<br />
<br />
====Competent Cell (CP919-Cph8)====<br />
#Day1. Streak out the E.coli strain on an LBM plate (with kanamycin antibiotic) to isolate colonies and incubate at 37℃ overnight (16-20 hours).<br />
#Day2. Select a single colony and inoculate 10 ml sterile LBM Grow overnight (16-20 hours) in a 37℃ shaker incubator.<br />
#Day3. Add 2ml overnight culture to 250ml flask which with 100 ml sterile LBM.<br />
#Grow the cultures to OD600 = 0.2~0.4 (incubate about 75~90 min.).<br />
#Centrifuge, 4℃,3000 r.p.m,10 min.<br />
#Discard the supernatant and mix the cell pellet with 10ml FSB.<br />
#Keep the cells on ice for 3~4 hours.<br />
#Centrifuge, 4℃,3000 r.p.m,10 min.<br />
#Discard the supernatant and mix the cell pellet with 5ml FSB.<br />
#Pipet 200μl of the cell suspension into sterile 1.5ml eppendorf. Freeze these tubes on N2 (aq.) and then transfer them to -80℃ freezer.<br />
<br />
<br />
====Real Time PCR====<br />
<br />
<br />
1. Take ice for preparing.<br />
<br />
2. dilution of concentration:<br />
<br />
:'''2.1'''<br />
:template DNA(Omp R):500 times diluted <br />
:499μl ddH2O + 1μl omp R=500μl<br />
<br />
:'''2.2'''<br />
:primer forward & primer reverse(p88):10 times diluted<br />
:10μl p88 primer forward+ 90μl ddH2O<br />
<br />
3. Materials:<br />
<br />
Template DNA(10ng/μl) 5<br />
10× PCR buffer 2<br />
10× dNTP(2mM) 2<br />
forward primer(10μM) 0.5<br />
reverse primer(10μM) 0.5<br />
Pfu DNA polymerase(2Kb) 0.1<br />
PCR water 9.9<br />
_______________________________________<br />
Total 20 μl<br />
<br />
4. Step:<br />
<br />
Meacure template DNA 2μl<br />
Measure 10× PCR buffer 2μl<br />
Measure 10× dNTP 2μl<br />
Measure forward primer 0.5μl<br />
Measure reverse primer 0.5μl<br />
Measure Pfu DNA polymerase 0.1μl<br />
Follow this step ,puttng the reagent in the eppendorf.<br />
<br />
5. Use the PCR to amplify our product:PCR program<br />
<br />
<br />
:'''5.1'''<br />
:94℃ 30 seconds<br />
:60℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 9 times<br />
<br />
<br />
:'''5.2'''<br />
:94℃ 30 seconds<br />
:55℃ 30seconds<br />
:72℃ 2 minutes<br />
:Cycle 34 times<br />
:72℃ 10 minutes<br />
<br />
<br />
6. The PCR production separate by 1% agar.<br />
<br />
7. After separate using the EtBr to dye agar and exposing by UV.<br />
<br />
<br />
====T-A cloning protocol====<br />
<br />
<br />
Take an eppendorf,and add these one by one,after mix well,overnight 4℃.<br />
<br />
<br />
2x ligase buffer 7.5μl<br />
Insert(Aeq.-GFP) 5.5μl<br />
Vector(pGEM-T-easy) 1μl<br />
T4 DNA exp 3/12 1μl<br />
_________________________________<br />
total 15μl<br />
<br />
<br />
====Cloning PCR====<br />
#Add 10μl cell, centrifuge 14000 rpm, 10 min<br />
#Discard the supernatant<br />
#Add 500μl ddH20, Votex<br />
#Boiling 20min, 100℃<br />
#Add 5μl DNA, 10x dNTP 2μl, 10x Buffer 2μl, forward primer(10μM)0.5μl reverse primer(10μM)0.5μl, Taq DNA polymerase 0.1μl, ddH20<br />
#Use the PCR to amplify our product:PCR program<br />
<br />
95℃ 4 min <br />
94℃ 30seconds <br />
55℃ 40seconds <br />
72℃ 2 min<br />
Cycle 34 times<br />
25℃ 2 min<br />
7. The PCR production separate by 1% agar. <br />
<br />
8. After separate using the EtBr to dye agar and exposing by UV.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ProjectTeam:TzuChiU Formosa/Project2009-10-20T12:46:01Z<p>TAI: /* Project */</p>
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<br />
==Project==<br />
<br />
No more electricity, No more pollution; The Midnight Apollo!<br />
<br />
<br />
In Taipei Taiwan alone, on average 768 mega watt of electricity for streetlamps was consumed every night (Department Public works, Taipei City government). It is beyond our imagination how much energy was consumed for the whole world. In Taiwan there are 9 power stations generated by coal, and produce 269.1 million tons of CO2 every year (based on data from CARMA), Power stations are major causes for global warming. Therefore, we would like to create” biolight” that can reduce CO2 generation and attenuate degree of global warming.<br />
<br />
<br />
We plan to create a new organism that doesn’t need electricity and cause no pollution, we will call “The Midnight Apollo”. “The Midnight Apollo” will be turned on when surrounding area turns dark and will be turned off automatically when the environment is bright. The idea is based on two systems, Cph8 and aequorin/GFP. The Cph8 is regulated by the visible light that can activate protein translation of an illuminating system. This illuminating system use aequorin/GFP to light up the environment.<br />
<br />
<br />
We hope The Midnight Apollo could be applied in producing energy-saved streetlamp, emergency lighting, or may even be used as searchlight.<br />
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<hr />
<div>[[Image:Tcu.jpg|left|caption]]<br />
<br />
<br />
''In 1966, Master Cheng Yen established Buddhist Tzu Chi Foundation which practices the philosophy of relieving suffering, giving happiness, helping the poor, and educating the rich. Nowadays, Tzu Chi people around the world reach out to the disaster areas, and relieve the suffering and agony of people in need.''<br />
<br />
''Tzu Chi University was in the eastern region of Taiwan which was established by Dharma Master Cheng Yen to raise the level of health care and education and to educate students to do good deeds and help others. Furthermore, our university expects the students to continue carrying out even after they graduate.''<br />
<br />
''With a spacious campus and green grass everywhere, Tzu Chi University provides well- equipped teaching facilities and a comfortable and graceful living environment, sponsors various student clubs, and provides a wonderful research atmosphere. This is a university that blends professionalism with humanitarianism.''<br />
<br />
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<br />
== '''Who we are''' ==<br />
<br />
<br />
[[Image:DSC03917 800.JPG]] <br />
<br />
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<br />
<br />
<br />
.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/NotebookTeam:TzuChiU Formosa/Notebook2009-10-19T16:42:48Z<p>TAI: /* Notebook */</p>
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<br />
==Notebook==<br />
'''Meeting'''<br />
<br />
<br />
<br />
'''2009.03''' Poster found<br />
<br />
People of different fields found Poster.<br />
<br />
<br />
'''2009.03.14''' Experience sharing<br />
<br />
National Yang-Ming University 2008 IGEM team shared<br />
Experience to TZU-CHI university.<br />
<br />
<br />
'''2009.05.07''' Firt meeting<br />
<br />
Introduction of IGEM and adviser.<br />
<br />
<br />
'''2009.05.15''' 2nd meeting<br />
<br />
Number of member identify.<br />
<br />
<br />
'''2009.05.28''' 3th meeting<br />
<br />
Idea report, chose 2 idea from 12,and experimented.<br />
<br />
<br />
'''2009.07~2009.09''' English class<br />
<br />
Enhanced the English proficiency on Wednesday,Thursday.<br />
<br />
<br />
<br />
'''2009.08.09''' Japanese friends visited<br />
<br />
Set up friendship with University of Tokyo, Chiba University, Japan.<br />
<br />
<br />
'''2009.08.31''' 4th meeting<br />
<br />
Progress report of the two groups.<br />
<br />
<br />
'''2009.09.14''' 5th meeting<br />
<br />
chose 1 team from 2 teams.<br />
<br />
<br />
'''2009.09.19''' 6th meeting<br />
<br />
Data of Searching report.<br />
<br />
<br />
'''2009.09.23''' 7th meeting<br />
<br />
Data of Searching report and Determined the direction of the experiment.<br />
<br />
<br />
'''2009.09.30''' 8th meeting with adviser<br />
<br />
Data of Searching and the direction of the experiment report to adviser.<br />
<br />
<br />
'''2009.10.2''' 9th meeting<br />
<br />
The distribution of staff that working and upload the team to wiki.<br />
<br />
<br />
'''2009.10.11''' 10th meeting<br />
<br />
Progress report and uplosd project, protocal to wiki.<br />
<br />
<br />
'''2009.10.15''' 11th meeting<br />
<br />
Progress report.<br />
<br />
<br />
'''2009.10.17''' 12th meeting<br />
<br />
Upload result, discussion, notebook to wiki.<br />
<br />
<br />
'''2009.10.20''' 13th meeting<br />
<br />
Progress report and modification of wiki with adviser.<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''experiment'''<br />
<br />
<br />
<br />
'''2009.10.06''' OmpC promoter transformation.<br />
<br />
<br />
'''2009.10.09''' Streak out OmpC promoter,pSB1A2 plasim extraction.<br />
<br />
<br />
'''2009.10.11''' OmpC stock.<br />
<br />
<br />
'''2009.10.12''' T-A cloning.<br />
<br />
<br />
'''2009.10.13''' T-Atransformation.<br />
<br />
<br />
'''2009.10.14''' Cloning PCR.<br />
<br />
<br />
'''2009.10.16''' T-A cloning.<br />
<br />
<br />
'''2009.10.20''' Digestion, Gel extration<br />
<br />
<br />
'''2009.10.21''' Gel extration<br />
<br />
<br />
<br />
<br />
.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/NotebookTeam:TzuChiU Formosa/Notebook2009-10-19T16:34:57Z<p>TAI: </p>
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<br />
==Notebook==<br />
'''Meeting'''<br />
<br />
<br />
<br />
'''2009.03''' Poster found<br />
<br />
People of different fields found Poster.<br />
<br />
<br />
'''2009.03.14''' Experience sharing<br />
<br />
National Yang-Ming University 2008 IGEM team shared<br />
Experience to TZU-CHI university.<br />
<br />
<br />
'''2009.05.07''' Firt meeting<br />
<br />
Introduction of IGEM and adviser.<br />
<br />
<br />
'''2009.05.15''' 2nd meeting<br />
<br />
Number of member identify.<br />
<br />
<br />
'''2009.05.28''' 3th meeting<br />
<br />
Idea report, chose 2 idea from 12,and experimented.<br />
<br />
<br />
'''2009.07~2009.09''' English class<br />
<br />
Enhanced the English proficiency on Wednesday,Thursday.<br />
<br />
<br />
<br />
'''2009.08.09''' Japanese friends visited<br />
<br />
Set up friendship with University of Tokyo, Chiba University, Japan.<br />
<br />
<br />
'''2009.08.31''' 4th meeting<br />
<br />
Progress report of the two groups.<br />
<br />
<br />
'''2009.09.14''' 5th meeting<br />
<br />
chose 1 team from 2 teams.<br />
<br />
<br />
'''2009.09.19''' 6th meeting<br />
<br />
Data of Searching report.<br />
<br />
<br />
'''2009.09.23''' 7th meeting<br />
<br />
Data of Searching report and Determined the direction of the experiment.<br />
<br />
<br />
'''2009.09.30''' 8th meeting with adviser<br />
<br />
Data of Searching and the direction of the experiment report to adviser.<br />
<br />
<br />
'''2009.10.2''' 9th meeting<br />
<br />
The distribution of staff that working and upload the team to wiki.<br />
<br />
<br />
'''2009.10.11''' 10th meeting<br />
<br />
Progress report and uplosd project, protocal to wiki.<br />
<br />
<br />
'''2009.10.15''' 11th meeting<br />
<br />
Progress report.<br />
<br />
<br />
'''2009.10.17''' 12th meeting<br />
<br />
Upload result, discussion, notebook to wiki.<br />
<br />
<br />
'''2009.10.20''' 13th meeting<br />
<br />
Progress report and modification of wiki with adviser.<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''experiment'''<br />
<br />
<br />
<br />
'''2009.10.06''' OmpC promoter transformation.<br />
<br />
<br />
'''2009.10.09''' Streak out OmpC promoter,pSB1A2 plasim extraction.<br />
<br />
<br />
'''2009.10.11''' OmpC stock.<br />
<br />
<br />
'''2009.10.12''' T-A cloning.<br />
<br />
<br />
'''2009.10.13''' T-Atransformation.<br />
<br />
<br />
'''2009.10.14''' Cloning PCR.<br />
<br />
<br />
'''2009.10.16''' T-A cloning.<br />
<br />
<br />
<br />
<br />
.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/backgroundTeam:TzuChiU Formosa/background2009-10-19T16:34:04Z<p>TAI: </p>
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<br />
==Background==<br />
<br />
<br />
====The Light Sensor====<br />
.<br />
<br />
'''Cph8'''<br />
<br />
<br />
The light sensor is an artificial gene regulation system, which can detect visible light. For our project the light sensor will initialize the signal cascade than activated the reporter gene(aeguorin-GFP).<br />
<br />
<br />
Plants and some bacteria use phytochromes to control photosynthesis. The light-sensitive protein pigment from cyanobacterium Synechocystis sp. PCC6803 consist a two-composed protein. The one component is a cyanobacterial phytochrome 1(Cph1) which is membrane bound sensor that responds to light. The other component is phycocyanobilin(PCB)which is iron that bind to the Cph1 and make the component to sensitize far red light. The photoreceptor are not find in Esherichia coil(E.coil), so the creational a light sensor was produced in 2005 by Austin iGEM team.<br />
The Austin iGEM team created the Cph8 which transform to Cp919. Cp919 is an EnvZ knockout E.coil strain that domain has been well studied. The chimaera Cph8 was a fusion protein which had cyanobacterial phytochrome 1(Cph1)with E.coil intracellular histidine kinase domain(EnvZ). They also insert two phycocyanobilin -biosynthesis gene(ho1 and pcyA)into [[User:Vul3|Vul3]]that it convert heam into phycocyanobilin. The part of the photoreceptor responds to light after PCB bind to cph1. The response-regulator affect transcription by the OmpR-dependent OmcC promoter and the aeguorin-GFP repoter. The cph8 is a bridge between light sensor and gene regulation.<br />
<br />
<br />
For our project, we also transform the reporter gene(aeguorin-GFP)to Cp919.In the light, the cph8 had little kinase activity and the OmpR remained unphosphorylated, and reporter gene was not expressed. In the dark, the cph8 had activity, high levels of OmpR phosphorylated, OmpR-p bind to ompC promoter, resulting in high aeguorin-GFP production.<br />
<br />
<br />
====The Light Expression====<br />
.<br />
<br />
'''Aequorin and GFP'''<br />
<br />
<br />
[[Image:Aequorin_1EJ3.png|thumb|200px|Aequorin]]<br />
[[Image:GFP_structure.png|thumb|200px|GFP]]<br />
There are many marine organism have fluorescence. But have you ever recognized one thing? How could it happen? The answer is Aequorin and GFP. <br />
<br />
<br />
Aequorin and GFP(Green Fluorescent Protein)has presented for more than one hundred millions years and discovered only in one jellyfish species-Aequorea Victoria. And when Ca2+ ions bind on aequorin, which will inducing a glowing blue light and GFP will raise to excited state and emit green fluorescence; it has a major excitation peak of wavelength at 395nm, the second peak is at 475nm and emission peak is at 509nm. <br />
<br />
<br />
Aequorin is composed of two distinct units, the apoprotein: apoaequorin and the prosthetic group: coelenterazine (a luciferin).<br />
<br />
<br />
Ca2+ is an indicator of Aequorin. When it binds on Ca2+, the prosthetic group will be oxidized. The coelenterazine converts into the excited state coelenteramide, and then relaxes to the ground state. Then Aequorin can emit the blue light (wavelenght= 469 nm) and supply energy for GFP.<br />
<br />
<br />
The structure of GFP is beta barrel, it constituted beta sheets with alpha helix and chromophore is located in the middle of the beta barrel which is a fluorescence of GFP.<br />
<br />
<br />
With this knowledge, Both of GFP will be a biosensor to reporter of expression in the cell or molecule biology; in our research we want GFP has strong enough to illume and to replace streetlamp and hope one day it can substitute the whole world daylight lamp.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/backgroundTeam:TzuChiU Formosa/background2009-10-19T16:32:04Z<p>TAI: </p>
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<br />
<br />
==Background==<br />
<br />
<br />
====The Light Sensor====<br />
<br />
<br />
'''Cph8'''<br />
<br />
<br />
The light sensor is an artificial gene regulation system, which can detect visible light. For our project the light sensor will initialize the signal cascade than activated the reporter gene(aeguorin-GFP).<br />
<br />
<br />
Plants and some bacteria use phytochromes to control photosynthesis. The light-sensitive protein pigment from cyanobacterium Synechocystis sp. PCC6803 consist a two-composed protein. The one component is a cyanobacterial phytochrome 1(Cph1) which is membrane bound sensor that responds to light. The other component is phycocyanobilin(PCB)which is iron that bind to the Cph1 and make the component to sensitize far red light. The photoreceptor are not find in Esherichia coil(E.coil), so the creational a light sensor was produced in 2005 by Austin iGEM team.<br />
The Austin iGEM team created the Cph8 which transform to Cp919. Cp919 is an EnvZ knockout E.coil strain that domain has been well studied. The chimaera Cph8 was a fusion protein which had cyanobacterial phytochrome 1(Cph1)with E.coil intracellular histidine kinase domain(EnvZ). They also insert two phycocyanobilin -biosynthesis gene(ho1 and pcyA)into [[User:Vul3|Vul3]]that it convert heam into phycocyanobilin. The part of the photoreceptor responds to light after PCB bind to cph1. The response-regulator affect transcription by the OmpR-dependent OmcC promoter and the aeguorin-GFP repoter. The cph8 is a bridge between light sensor and gene regulation.<br />
<br />
<br />
For our project, we also transform the reporter gene(aeguorin-GFP)to Cp919.In the light, the cph8 had little kinase activity and the OmpR remained unphosphorylated, and reporter gene was not expressed. In the dark, the cph8 had activity, high levels of OmpR phosphorylated, OmpR-p bind to ompC promoter, resulting in high aeguorin-GFP production.<br />
<br />
<br />
====The Light Expression====<br />
<br />
<br />
'''Aequorin and GFP'''<br />
<br />
<br />
[[Image:Aequorin_1EJ3.png|thumb|200px|Aequorin]]<br />
[[Image:GFP_structure.png|thumb|200px|GFP]]<br />
There are many marine organism have fluorescence. But have you ever recognized one thing? How could it happen? The answer is Aequorin and GFP. <br />
<br />
<br />
Aequorin and GFP(Green Fluorescent Protein)has presented for more than one hundred millions years and discovered only in one jellyfish species-Aequorea Victoria. And when Ca2+ ions bind on aequorin, which will inducing a glowing blue light and GFP will raise to excited state and emit green fluorescence; it has a major excitation peak of wavelength at 395nm, the second peak is at 475nm and emission peak is at 509nm. <br />
<br />
<br />
Aequorin is composed of two distinct units, the apoprotein: apoaequorin and the prosthetic group: coelenterazine (a luciferin).<br />
<br />
<br />
Ca2+ is an indicator of Aequorin. When it binds on Ca2+, the prosthetic group will be oxidized. The coelenterazine converts into the excited state coelenteramide, and then relaxes to the ground state. Then Aequorin can emit the blue light (wavelenght= 469 nm) and supply energy for GFP.<br />
<br />
<br />
The structure of GFP is beta barrel, it constituted beta sheets with alpha helix and chromophore is located in the middle of the beta barrel which is a fluorescence of GFP.<br />
<br />
<br />
With this knowledge, Both of GFP will be a biosensor to reporter of expression in the cell or molecule biology; in our research we want GFP has strong enough to illume and to replace streetlamp and hope one day it can substitute the whole world daylight lamp.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/NotebookTeam:TzuChiU Formosa/Notebook2009-10-19T16:27:39Z<p>TAI: /* Notebook */</p>
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==Notebook==<br />
'''Meeting'''<br />
<br />
<br />
<br />
'''2009.03''' Poster found<br />
<br />
People of different fields found Poster.<br />
<br />
<br />
'''2009.03.14''' Experience sharing<br />
<br />
National Yang-Ming University 2008 IGEM team shared<br />
Experience to TZU-CHI university.<br />
<br />
<br />
'''2009.05.07''' Firt meeting<br />
<br />
Introduction of IGEM and adviser.<br />
<br />
<br />
'''2009.05.15''' 2nd meeting<br />
<br />
Number of member identify.<br />
<br />
<br />
'''2009.05.28''' 3th meeting<br />
<br />
Idea report, chose 2 idea from 12,and experimented.<br />
<br />
<br />
'''2009.07~2009.09''' English class<br />
<br />
Enhanced the English proficiency on Wednesday,Thursday.<br />
<br />
<br />
<br />
'''2009.08.09''' Japanese friends visited<br />
<br />
Set up friendship with University of Tokyo, Chiba University, Japan.<br />
<br />
<br />
'''2009.08.31''' 4th meeting<br />
<br />
Progress report of the two groups.<br />
<br />
<br />
'''2009.09.14''' 5th meeting<br />
<br />
chose 1 team from 2 teams.<br />
<br />
<br />
'''2009.09.19''' 6th meeting<br />
<br />
Data of Searching report.<br />
<br />
<br />
'''2009.09.23''' 7th meeting<br />
<br />
Data of Searching report and Determined the direction of the experiment.<br />
<br />
<br />
'''2009.09.30''' 8th meeting with adviser<br />
<br />
Data of Searching and the direction of the experiment report to adviser.<br />
<br />
<br />
'''2009.10.2''' 9th meeting<br />
<br />
The distribution of staff that working and upload the team to wiki.<br />
<br />
<br />
'''2009.10.11''' 10th meeting<br />
<br />
Progress report and uplosd project, protocal to wiki.<br />
<br />
<br />
'''2009.10.15''' 11th meeting<br />
<br />
Progress report.<br />
<br />
<br />
'''2009.10.17''' 12th meeting<br />
<br />
Upload result, discussion, notebook to wiki.<br />
<br />
<br />
'''2009.10.20''' 13th meeting<br />
<br />
Progress report and modification of wiki with adviser.<br />
<br />
<br />
<br />
<br />
<br />
<br />
'''experiment'''<br />
<br />
<br />
<br />
'''2009.10.06''' OmpC promoter transformation.<br />
<br />
<br />
'''2009.10.09''' Streak out OmpC promoter,pSB1A2 plasim extraction.<br />
<br />
<br />
'''2009.10.11''' OmpC stock.<br />
<br />
<br />
'''2009.10.12''' T-A cloning.<br />
<br />
<br />
'''2009.10.13''' T-Atransformation.<br />
<br />
<br />
'''2009.10.14''' Cloning PCR.<br />
<br />
<br />
'''2009.10.16''' T-A cloning.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/ResultTeam:TzuChiU Formosa/Result2009-10-19T16:14:58Z<p>TAI: /* Result */</p>
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==Result==<br />
<gallery><br />
Image:Result_f1a.jpg|a<br />
Image:Result_f1b.jpg|b<br />
Image:Result_f1c.jpg|c<br />
</gallery><br />
'''Figure.1(a.)SB1A3 cloning PCR gel image. start with left to right part:(b.)100b.p. marker,(PS)PSB1A3,(PS)PSB1A3,(N)Negative control。(b.)the plate is PSB1A3 transformation into DH5α’s single colony。(c.)ompC plasmid gel image。start with left to right part:(M)100b.p. marker,(C)ompC,(C)ompC。'''<br />
<br />
<br />
According to cloning PCR experiment we could make sure that PSB1A3 contained OmpC, and the size of OmpC was about 108 base pair. From Fig.1-a we could know that template was placed at 108 bp, which confirmed that PSB1A3 really contained OmpC promoter. After transforming PSB1A3 into DH5a , culturing and spirting; From Fig1-b we can see the growing of single colony. Picking two similar size of single colonies, after being cultured in 37℃ and being amplified, draw out PSB1A3 plasmid and run gel electrophoresis to check if the transformation was succeed or not. The PSB1A3 size was about 2.75kb; Fig1-c shows plasmid was at 2.75kb, so it was confirmed that PSB1A3 had been successfully transformed into DH5a. <br />
<br />
<br />
<gallery><br />
Image:Result_f2a.jpg|a<br />
Image:Result_f2b.jpg|b<br />
Image:Result_f2c.jpg|c<br />
</gallery><br />
'''Figure.2(a.)Aquorin-GFP sequence after purify gel image。Left:(M)100b.p. marker,(AG)Aquorin-GFP。(b.)After TA cloning transformation into DH5α,spirt and run the blue white selection.(c.)Aequorin-GFP cloning PCR gel image。Start with left to right:(M)100b.p.marker,the other(A-G)Aquorin-GFP。<br />
'''<br />
<br />
<br />
Because of Aquorin-GFP didn't know the purify and concentration;Through PCR, we run the purify step to increasing the purify. The Aquorin-GFP size was about 2000bp;Fig.2-a show that template was placed at 2000 bp,so it confirm that suecssfully purify the Aquorin-GFP. And took the successful purify Aquorin-GFP to run the TA cloning, let Aquorin-GFP could ligate to Pgem-T-easy plasmid. After transformation into DH5a, then spirting the plate contain Ampicilin, 100ul IPTG, 50 ul X-gal. As the result of blue white selection was white single colony which was we expect.Fig2-b: Show that the white single colony was the successful form. And we picked from the two closing size single colony after put at the 37℃ and amplify, then run cloning PCR show that Aquorin-GFP size was about 2000bp. Fig.2-c show that the sequence was placed at 2000bp,so it confirm Aquorin-GFP had been successfully ligate to Pgem-T-easy plasmid。<br />
<br />
<br />
<gallery><br />
Image:Result_f3a.jpg|a<br />
</gallery><br />
'''Figure.3(a.)PSB1A3 digestion gel image.Start with left to right:(M)100b.p.marker, (None)None cut PSB1A3, (None)None cut PSB1A3, (cut)cut PSB1A3.'''<br />
<br />
<br />
When we confirm the PSB1A3 contain OmpC. And we amplify it, used Pst1 to cutting off the sequences and run the gel electrophoresis. Due to the cutting form plasmid will change circular to linear form caused molecular weight increasing so the gel electrophoresis speed will slow down and stop at the upper strata show at the Fig.3-a.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/DiscussionTeam:TzuChiU Formosa/Discussion2009-10-19T16:05:41Z<p>TAI: /* Discussion */</p>
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==Discussion==<br />
<br />
<br />
<br />
<br />
'''digestion'''<br />
<br />
The cause of unsuccessful digestion might be the few data after progress A-G PCR, or the concentration was not high enough. <br />
<br />
'''TA cloning'''<br />
<br />
Why using TA cloning? Because of its connecting way was “blunt end”, and that improves ligation’s efficiency. Furthermore, it is no need to do the digestion. But the main defect of this technique is that it doesn’t have concentrated ability, so in the next step it is still risky to disperse. Our experiment was also having other insert in it, which cause the misconnecting to the wrong site. In order to reduce the risk we have to raise the insert’s purity or take more single colonies to increasing the purity.<br />
<br />
<br />
'''PSBlA3'''<br />
<br />
When we started to transformations, we discovered that the colony still couldn’t be cultured. And we’ve figured out three reasons. First, at the heat shock time stage we using four times to test : 45(sec), 60(sec), 90(sec), 120(sec). And we found out that 60(sec) has better result than others, but compared to the paper’s result, it was still not going so well. So we speculated that it was also affected by the time when it was placed on the ice. Second, the time of the mixture of competent cell and plasmid on the ice might be too long (over 30 minutes). We have found in some protocol that only put it for five minutes. So we change it into five minutes, and the effect was very great. The last reason is the competent cell might be placed for a while which decreased the efficiency.<br />
<br />
Due to the A-G sequence was supplied by France, the vector concentration and purity was unknown by us. We need to go through a series of purifying steps. After finish the A-G PCR, we discovered that it still included some unclear band. When we finish the whole purifying progress, it still had some unknown band. For the better purity and higher A-G concentration, we chose TA cloning and cloning PCR. Because of the previous experiment, TA cloning has been interfered by the unclear band. That cause TA cloning binding to the wrong insert. So we pick the other plate, and this time it was very lucky that we successfully got the correct result.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/DiscussionTeam:TzuChiU Formosa/Discussion2009-10-19T16:04:16Z<p>TAI: /* Discussion */</p>
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==Discussion==<br />
<br />
<br />
<br />
<br />
'''digestion'''<br />
<br />
The cause of unsuccessful digestion might be the few data after progress A-G PCR, or the concentration was not high enough. <br />
<br />
'''TA cloning'''<br />
<br />
<br />
Why using TA cloning? Because of its connecting way was “blunt end”, and that improves ligation’s efficiency. Furthermore, it is no need to do the digestion. But the main defect of this technique is that it doesn’t have concentrated ability, so in the next step it is still risky to disperse. Our experiment was also having other insert in it, which cause the misconnecting to the wrong site. In order to reduce the risk we have to raise the insert’s purity or take more single colonies to increasing the purity.<br />
<br />
<br />
'''PSBlA3'''<br />
<br />
<br />
When we started to transformations, we discovered that the colony still couldn’t be cultured. And we’ve figured out three reasons. First, at the heat shock time stage we using four times to test : 45(sec), 60(sec), 90(sec), 120(sec). And we found out that 60(sec) has better result than others, but compared to the paper’s result, it was still not going so well. So we speculated that it was also affected by the time when it was placed on the ice. Second, the time of the mixture of competent cell and plasmid on the ice might be too long (over 30 minutes). We have found in some protocol that only put it for five minutes. So we change it into five minutes, and the effect was very great. The last reason is the competent cell might be placed for a while which decreased the efficiency.<br />
<br />
<br />
Due to the A-G sequence was supplied by France, the vector concentration and purity was unknown by us. We need to go through a series of purifying steps. After finish the A-G PCR, we discovered that it still included some unclear band. When we finish the whole purifying progress, it still had some unknown band. For the better purity and higher A-G concentration, we chose TA cloning and cloning PCR. Because of the previous experiment, TA cloning has been interfered by the unclear band. That cause TA cloning binding to the wrong insert. So we pick the other plate, and this time it was very lucky that we successfully got the correct result.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/NotebookTeam:TzuChiU Formosa/Notebook2009-10-18T03:44:10Z<p>TAI: </p>
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==Notebook==<br />
'''Meeting'''<br />
<br />
<br />
<br />
'''2009.03''' Poster found<br />
<br />
People of different fields found Poster.<br />
<br />
<br />
'''2009.03.14''' Experience sharing<br />
<br />
National Yang-Ming University 2008 IGEM team shared<br />
Experience to TZU-CHI university.<br />
<br />
<br />
'''2009.05.07'''' Firt meeting<br />
<br />
Introduction of IGEM and adviser.<br />
<br />
<br />
'''2009.05.15''' 2nd meeting<br />
<br />
Number of member identify.<br />
<br />
<br />
'''2009.05.28''' 3th meeting<br />
<br />
Idea report, chose 2 idea from 12,and experimented.<br />
<br />
<br />
'''2009.07~2009.09''' English class<br />
<br />
Enhanced the English proficiency on Wednesday,Thursday.<br />
<br />
<br />
<br />
'''2009.08.09''' Japanese friends visited<br />
<br />
Set up friendship with University of Tokyo, Chiba University, Japan.<br />
<br />
<br />
'''2009.08.31''' 4th meeting<br />
<br />
Progress report of the two groups.<br />
<br />
<br />
'''2009.09.14''' 5th meeting<br />
<br />
chose 1 team from 2 teams.<br />
<br />
<br />
'''2009.09.19''' 6th meeting<br />
<br />
Data of Searching report.<br />
<br />
<br />
'''2009.09.23''' 7th meeting<br />
<br />
Data of Searching report and Determined the direction of the experiment.<br />
<br />
<br />
'''2009.09.30''' 8th meeting with adviser<br />
<br />
Data of Searching and the direction of the experiment report to adviser.<br />
<br />
<br />
''2009.10.2''' 9th meeting<br />
<br />
The distribution of staff that working.<br />
<br />
<br />
'''2009.10.11''' 10th meeting<br />
<br />
Progress report.<br />
<br />
<br />
'''2009.10.15''' 11th meeting<br />
<br />
Progress report.<br />
<br />
<br />
'''experiment'''<br />
<br />
<br />
<br />
'''2009.10.06''' OmpC promoter transformation.<br />
<br />
<br />
'''2009.10.09''' Streak out OmpC promoter,pSB1A2 plasim extraction.<br />
<br />
<br />
'''2009.10.11''' OmpC stock.<br />
<br />
<br />
'''2009.10.12''' T-A cloning.<br />
<br />
<br />
'''2009.10.13''' T-Atransformation.<br />
<br />
<br />
'''2009.10.14''' Cloning PCR.<br />
<br />
<br />
'''2009.10.16''' T-A cloning.</div>TAIhttp://2009.igem.org/Team:TzuChiU_Formosa/NotebookTeam:TzuChiU Formosa/Notebook2009-10-18T03:34:16Z<p>TAI: </p>
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==Notebook==<br />
'''Meeting'''<br />
<br />
<br />
<br />
'''2009.03''' Poster found<br />
<br />
People of different fields found Poster.<br />
<br />
<br />
'''2009.03.14''' Experience sharing<br />
<br />
National Yang-Ming University 2008 IGEM team shared<br />
Experience to TZU-CHI university.<br />
<br />
<br />
'''2009.05.07'''' Firt meeting<br />
<br />
Introduction of IGEM and adviser.<br />
<br />
<br />
'''2009.05.15''' 2nd meeting<br />
<br />
Number of member identify.<br />
<br />
<br />
'''2009.05.28''' 3th meeting<br />
<br />
Idea report, chose 2 idea from 12,and experimented.<br />
<br />
<br />
'''2009.07~2009.09''' English class<br />
<br />
Enhanced the English proficiency on Wednesday,Thursday.<br />
<br />
<br />
<br />
'''2009.08.09''' Japanese friends visited<br />
<br />
Set up friendship with University of Tokyo, Chiba University, Japan.<br />
<br />
<br />
'''2009.08.31''' 4th meeting<br />
<br />
Progress report of the two groups.<br />
<br />
<br />
'''2009.09.14''' 5th meeting<br />
<br />
chose 1 team from 2 teams.<br />
<br />
<br />
'''2009.09.23''' 6th meeting<br />
<br />
<br />
<br />
'''experiment'''<br />
<br />
<br />
<br />
'''2009.10.06''' OmpC promoter transformation.<br />
<br />
<br />
'''2009.10.09''' Streak out OmpC promoter,pSB1A2 plasim extraction.<br />
<br />
<br />
'''2009.10.11''' OmpC stock.<br />
<br />
<br />
'''2009.10.12''' T-A cloning.<br />
<br />
<br />
'''2009.10.13''' T-Atransformation.<br />
<br />
<br />
'''2009.10.14''' Cloning PCR.<br />
<br />
<br />
'''2009.10.16''' T-A cloning.</div>TAI