Team:TzuChiU Formosa/Notebook/photopaper
From 2011.igem.org
(→2011.09.07) |
(→Protocols) |
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==='''2011.09.07'''=== | ==='''2011.09.07'''=== | ||
- | [[File:Catt.gif|right|250px|caption]] | + | <br>[[File:Catt.gif|right|250px|caption]] |
'''Rhodobacter rudrum medium''' | '''Rhodobacter rudrum medium''' | ||
<br> | <br> | ||
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<br> | <br> | ||
<br> | <br> | ||
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==='''2011.09.08-13'''=== | ==='''2011.09.08-13'''=== | ||
<br> | <br> | ||
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<br>1.50ml LB+500μl CHLORAMPHENICOL | <br>1.50ml LB+500μl CHLORAMPHENICOL | ||
<br>2.37℃, overnight (14-16hrs) | <br>2.37℃, overnight (14-16hrs) | ||
+ | <br> | ||
<br> | <br> | ||
<br> | <br> | ||
==='''2011.09.09'''=== | ==='''2011.09.09'''=== | ||
+ | <br> | ||
'''Raise Gluconacetobacter hansenii''' | '''Raise Gluconacetobacter hansenii''' | ||
<br> | <br> | ||
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<br> | <br> | ||
==='''2011.09.10-11'''=== | ==='''2011.09.10-11'''=== | ||
+ | <br> | ||
'''Digestion check of DNA''' | '''Digestion check of DNA''' | ||
<br> | <br> | ||
<br>[PSB1C3/EcoRI] | <br>[PSB1C3/EcoRI] | ||
<br> | <br> | ||
- | DNA 500ng<br> | + | <br>DNA 500ng<br> |
- | 10×buffer 5μl<br> | + | <br>10×buffer 5μl<br> |
- | BSA 5μl<br> | + | <br>BSA 5μl<br> |
- | EcoRⅠ 1μl<br> | + | <br>EcoRⅠ 1μl<br> |
- | ddH2O 29μl<br> | + | <br>ddH2O 29μl<br> |
- | _______________________________ | + | <br>_______________________________ |
<br> | <br> | ||
- | total 50μl<br> | + | <br>total 50μl<br> |
<br> | <br> | ||
<br> | <br> | ||
+ | <br>[pSB1C3/PstⅠ] | ||
<br> | <br> | ||
- | + | <br>DNA 500ng<br> | |
+ | <br>10×buffer 5μl<br> | ||
+ | <br>BSA 5μl<br> | ||
+ | <br>pstⅠ 1μl<br> | ||
+ | <br>ddH2O 29μl<br> | ||
+ | <br>_________________________________ | ||
<br> | <br> | ||
- | + | <br>total 50μl<br> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<br> | <br> | ||
- | |||
<br> | <br> | ||
- | [PSB1C3/EcoRⅠ+PstⅠ] | + | <br>[PSB1C3/EcoRⅠ+PstⅠ] |
<br> | <br> | ||
- | DNA 500ng<br> | + | <br>DNA 500ng<br> |
- | 10×buffer 5μl<br> | + | <br>10×buffer 5μl<br> |
- | BSA 5μl<br> | + | <br>BSA 5μl<br> |
- | EcoRⅠ 1μl<br> | + | <br>EcoRⅠ 1μl<br> |
- | pstⅠ 1μl<br> | + | <br>pstⅠ 1μl<br> |
- | + | <br>ddH2O 28μl<br> | |
- | + | <br>__________________________________ | |
- | <br> | + | |
- | + | ||
- | + | ||
<br> | <br> | ||
+ | <br>total 50μl<br> | ||
+ | <br>→37℃ for 30 mins | ||
<br> | <br> | ||
<br> | <br> | ||
'''Digestion of DNA''' | '''Digestion of DNA''' | ||
<br> | <br> | ||
- | [PSB1C3/EcoRⅠ+pstⅠ] | + | <br>[PSB1C3/EcoRⅠ+pstⅠ] |
+ | <br> | ||
+ | <br>DNA 10μl<br> | ||
+ | <br>10×buffer 5μl<br> | ||
+ | <br>BSA 5μl<br> | ||
+ | <br>EcoRⅠ 1μl<br> | ||
+ | <br>pstⅠ 1μl<br> | ||
+ | <br>ddH2O 28μl<br> | ||
+ | <br>____________________________________ | ||
+ | <br> | ||
+ | <br>total 50μl<br> | ||
+ | <br>→37℃ for 2 hrs | ||
+ | <br> | ||
+ | '''electroelution Purification''' | ||
+ | <br> | ||
+ | <br>PSB1C3 backbone | ||
<br> | <br> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
<br> | <br> | ||
- | |||
- | |||
<br> | <br> | ||
- | |||
- | |||
- | |||
==='''2011.09.14-24'''=== | ==='''2011.09.14-24'''=== | ||
- | |||
- | |||
- | |||
- | |||
- | |||
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- | |||
- | |||
- | |||
- | |||
<br> | <br> | ||
- | + | '''PCR''' | |
- | + | <br>[[File:Cats2.gif |right|404px|caption]] | |
- | + | <br>template DNA 1μl | |
- | + | <br>5×Buffer 4μl | |
- | + | <br>2.5μM dNTP 1.6μl | |
+ | <br>10μM F 1μl | ||
+ | <br>10μM R 1μl | ||
+ | <br>Taq 0.2μl | ||
+ | <br>ddH2O 8.8μl | ||
+ | <br>_______________________________ | ||
+ | <br> | ||
+ | <br>total 20μl | ||
+ | <br> | ||
+ | <br> | ||
+ | <br> | ||
==='''2011.09.21'''=== | ==='''2011.09.21'''=== | ||
+ | <br> | ||
'''Digestion of DNA''' | '''Digestion of DNA''' | ||
+ | <br> | ||
<br>[acsAB/ XbaⅠ+SpeⅠ] | <br>[acsAB/ XbaⅠ+SpeⅠ] | ||
+ | <br> | ||
<br>DNA 10μl | <br>DNA 10μl | ||
<br>10×buffer 5μl | <br>10×buffer 5μl | ||
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<br>____________________________ | <br>____________________________ | ||
<br> | <br> | ||
- | total 50μl | + | <br>total 50μl |
<br>→37℃ for 16 hr | <br>→37℃ for 16 hr | ||
- | + | <br> | |
'''Digestion of DNA''' | '''Digestion of DNA''' | ||
+ | <br> | ||
<br>[acsCD/XbaⅠ+SpeⅠ] | <br>[acsCD/XbaⅠ+SpeⅠ] | ||
+ | <br> | ||
<br>DNA 10μl | <br>DNA 10μl | ||
<br>10×buffer 5μl | <br>10×buffer 5μl | ||
Line 282: | Line 291: | ||
total 50μl | total 50μl | ||
<br>→37℃ for 16 hr | <br>→37℃ for 16 hr | ||
- | + | <br> | |
- | + | <br> | |
- | + | <br> | |
- | + | ||
==='''2011.09.22'''=== | ==='''2011.09.22'''=== | ||
'''Ligation of DNA''' | '''Ligation of DNA''' | ||
<br>[PSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]] | <br>[PSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]] | ||
+ | <br> | ||
<br>Vector 3μl | <br>Vector 3μl | ||
<br>Insert 14μl | <br>Insert 14μl | ||
Line 296: | Line 305: | ||
<br>________________________________ | <br>________________________________ | ||
<br> | <br> | ||
- | total 20μl | + | <br>total 20μl |
- | + | <br> | |
+ | <br> | ||
'''Ligation of DNA''' | '''Ligation of DNA''' | ||
+ | <br> | ||
<br>[PSB1A3-acsCD] | <br>[PSB1A3-acsCD] | ||
+ | <br> | ||
<br>Vector 3μl | <br>Vector 3μl | ||
<br>Insert 14μl | <br>Insert 14μl | ||
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<br>_________________________________ | <br>_________________________________ | ||
<br> | <br> | ||
- | total 20μl | + | <br>total 20μl |
- | + | <br> | |
+ | <br> | ||
+ | <br> | ||
==='''2011.09.23'''=== | ==='''2011.09.23'''=== | ||
- | + | <br> | |
'''PCR''' | '''PCR''' | ||
+ | <br> | ||
<br>PR0011 promoter 1μl | <br>PR0011 promoter 1μl | ||
<br>5×Buffer 4μl | <br>5×Buffer 4μl | ||
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<br>_____________________________________ | <br>_____________________________________ | ||
<br> | <br> | ||
- | total 20μl | + | <br>total 20μl |
- | + | <br> | |
- | + | <br> | |
- | + | <br> | |
- | + | ||
- | + | ||
==='''2011.09.24'''=== | ==='''2011.09.24'''=== | ||
- | + | <br> | |
'''Transformation of DNA''' | '''Transformation of DNA''' | ||
+ | <br> | ||
<br>PSB1C3-acsAB | <br>PSB1C3-acsAB | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
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'''Transformation of DNA''' | '''Transformation of DNA''' | ||
+ | <br> | ||
<br>PSB1A3-acsCD | <br>PSB1A3-acsCD | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
Line 338: | Line 353: | ||
'''Transformation of DNA''' | '''Transformation of DNA''' | ||
+ | <br> | ||
<br>PSB1C3-acsAB | <br>PSB1C3-acsAB | ||
<br>PSB1A3-acsCD | <br>PSB1A3-acsCD | ||
<br>Transform into E.coli | <br>Transform into E.coli | ||
<br>LB+Ampicillin+CHLORAMPHENICOL | <br>LB+Ampicillin+CHLORAMPHENICOL | ||
- | + | <br> | |
- | + | <br> | |
- | + | <br> | |
- | + | ||
- | + | ||
==='''2011.09.24'''=== | ==='''2011.09.24'''=== | ||
- | + | <br> | |
'''Ligation of DNA''' | '''Ligation of DNA''' | ||
<br>[PSB1C3-promoter] | <br>[PSB1C3-promoter] | ||
+ | <br> | ||
<br>Vector 3μl | <br>Vector 3μl | ||
<br>Insert 14μl | <br>Insert 14μl | ||
Line 358: | Line 373: | ||
<br>________________________________ | <br>________________________________ | ||
<br>total 20μl | <br>total 20μl | ||
+ | <br> |
Revision as of 15:12, 3 October 2011
Contents |
Photopaper
Meeting Notes
2011.02.24
Discussion:
- Team organization
- Brain storming
- paper made by bacteria with add-ons such as colors, fragrance, etc.
- "light up" the plants for replacing lamp posts.
2011.03.04
Discussion:
- Team advisory
- Brain storming
- Add-ons: colorful cellulose which produced by bacteria such as beta-carotene
- information exchange with iGEM 2009 Cambridge team
2011.03.14
Discussion:
- Task Allocation
- Brain storming
- Avatar - "light up" the plants by transfecting symbiotic bacteria which cloned into fluorescence gene
- Eco-friendly warmer - biotic thermal pad
2011.03.23
Discussion:
- Project : paperia
- Option 1 : Culture bacteria which has pigment gene
- Option 2 : Cellulose-producing bacteria secrete pigment into the medium
2011.03.24
Discussion:
- Exp. procedure:
- cloning of cellulose gene’s CDS
- the product should operate within E. coli.
2011.06.22
Discussion:
- Due to some unforseen reason, the team decided to change their project.
- New project: Biojenny
-economical and humane way to produce paper in large quantities.
-yeast to be our host
2011.07.01
Discussion:
- Freeze > grin > genome DNA isolation > Cloning = silk protein gene
2011.07.09
Discussion:
- the connections between 3 silk proteins : Fibl Fibh P25
- major proteins : H-chain, L-chain, P25
2011.07.15
Discussion:
- Due to limited resources and techniques required, the team decided to switch back to the previous project. Paper making !
- However it would be modified to be more innovative and creative.
2011.07.18
Discussion:
- Latest project : Photo paper
- cyanobacteria is designed as the host, cellulose made up of the glucose produced by cyanobacteria could be one of the main attraction of the project.
2011.07.23
Discussion:
- system modification to overcome the problems arises during preliminary round
- Biobricks from Tokyo 2010 team will be utilized
- regulator promoter in order to regulate the secretion of cellulose to solve the aggregation of the bacteria
2011.09.15
Genome miniprep
Gluconacetobacter hansenii
2011.09.18
Gel/PCR DNA extraction
Gluconacetobacter hansenii
Protocols
2011.09.07
Rhodobacter rudrum medium
K2HPO4 1g
NaCl 0.5g
FeSO4.7H2O 0.01g
CaCl2 0.02g
MnCl2.4H2O 0.002g
MgSO4.7H2O 0.2g
NaMO2O4.2H2O 0.01g
ddH2O 998.258ml
________________________________________
1L →take100ml
+
Yeast Extrat 0.5g
Sodium malate
(Sodium succinate dibasic hexohydrate) 5g
NH4Cl 1g
ddH2O 893.5ml
_________________________________________________
1L
Raise E. coli(PSB1C3)
1.50ml LB+500μl CHLORAMPHENICOL
2.37℃, overnight (14-16hrs)
2011.09.08-13
Plasmid miniprep kit
PSB1C3 plasmid
Raise Rhodobacter rubrum
1.50ml LB+500μl CHLORAMPHENICOL
2.37℃, overnight (14-16hrs)
2011.09.09
Raise Gluconacetobacter hansenii
1.50ml LB+500μl CHLORAMPHENICOL
2.37℃, overnight (14-16hrs)
2011.09.10-11
Digestion check of DNA
[PSB1C3/EcoRI]
DNA 500ng
10×buffer 5μl
BSA 5μl
EcoRⅠ 1μl
ddH2O 29μl
_______________________________
total 50μl
[pSB1C3/PstⅠ]
DNA 500ng
10×buffer 5μl
BSA 5μl
pstⅠ 1μl
ddH2O 29μl
_________________________________
total 50μl
[PSB1C3/EcoRⅠ+PstⅠ]
DNA 500ng
10×buffer 5μl
BSA 5μl
EcoRⅠ 1μl
pstⅠ 1μl
ddH2O 28μl
__________________________________
total 50μl
→37℃ for 30 mins
Digestion of DNA
[PSB1C3/EcoRⅠ+pstⅠ]
DNA 10μl
10×buffer 5μl
BSA 5μl
EcoRⅠ 1μl
pstⅠ 1μl
ddH2O 28μl
____________________________________
total 50μl
→37℃ for 2 hrs
electroelution Purification
PSB1C3 backbone
2011.09.14-24
PCR
template DNA 1μl
5×Buffer 4μl
2.5μM dNTP 1.6μl
10μM F 1μl
10μM R 1μl
Taq 0.2μl
ddH2O 8.8μl
_______________________________
total 20μl
2011.09.21
Digestion of DNA
[acsAB/ XbaⅠ+SpeⅠ]
DNA 10μl
10×buffer 5μl
BSA 5μl
EcoRⅠ 1μl
pstⅠ 1μl
ddH2O 28μl
____________________________
total 50μl
→37℃ for 16 hr
Digestion of DNA
[acsCD/XbaⅠ+SpeⅠ]
DNA 10μl
10×buffer 5μl
BSA 5μl
EcoRⅠ 1μl
pstⅠ 1μl
ddH2O 28μl
__________________________________
total 50μl
→37℃ for 16 hr
2011.09.22
Ligation of DNA
[PSB1C3-acsAB]
Vector 3μl
Insert 14μl
ligase buffer 2μl
ligase 1μl
ddH2O -μl
________________________________
total 20μl
Ligation of DNA
[PSB1A3-acsCD]
Vector 3μl
Insert 14μl
ligase buffer 2μl
ligase 1μl
ddH2O -μl
_________________________________
total 20μl
2011.09.23
PCR
PR0011 promoter 1μl
5×Buffer 4μl
2.5μM dNTP 1.6μl
Taq 0.2μl
ddH2O 13.2μl
_____________________________________
total 20μl
2011.09.24
Transformation of DNA
PSB1C3-acsAB
Transform into E.coli
LB+CHLORAMPHENICOL
Transformation of DNA
PSB1A3-acsCD
Transform into E.coli
LB+Ampicillin
Transformation of DNA
PSB1C3-acsAB
PSB1A3-acsCD
Transform into E.coli
LB+Ampicillin+CHLORAMPHENICOL
2011.09.24
Ligation of DNA
[PSB1C3-promoter]
Vector 3μl
Insert 14μl
ligase buffer 2μl
ligase 1μl
ddH2O -μl
________________________________
total 20μl