Team:TzuChiU Formosa/Notebook/photopaper

From 2011.igem.org

(Difference between revisions)
(2011.09.07)
(Protocols)
Line 126: Line 126:
==='''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>
Line 158: Line 158:
<br>
<br>
<br>
<br>
-
 
==='''2011.09.08-13'''===
==='''2011.09.08-13'''===
<br>
<br>
Line 169: Line 168:
<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>
Line 180: Line 181:
<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>
-
[pSB1C3/PstⅠ]
+
<br>DNA             500ng<br>
 +
<br>10×buffer           5μl<br>
 +
<br>BSA               5μl<br>
 +
<br>pstⅠ              1μl<br>
 +
<br>ddH2O             29μl<br>                                             
 +
<br>_________________________________
<br>
<br>
-
DNA             500ng<br>
+
<br>total            50μl<br>
-
10×buffer           5μl<br>
+
-
BSA               5μl<br>
+
-
pstⅠ              1μl<br>
+
-
ddH2O            29μl<br>                                              
+
-
_________________________________
+
<br>
<br>
-
total             50μl<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>
-
ddH2O            28μl<br>            
+
<br>ddH2O            28μl<br>            
-
__________________________________
+
<br>__________________________________
-
<br>
+
-
total            50μl<br>
+
-
→37℃ for 30 mins
+
<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>
-
DNA               10μl<br>
 
-
10×buffer            5μl<br>
 
-
BSA               5μl<br>
 
-
EcoRⅠ              1μl<br>
 
-
pstⅠ               1μl<br>
 
-
ddH2O              28μl<br>
 
-
____________________________________
 
<br>
<br>
-
total              50μl<br>
 
-
→37℃ for 2 hrs
 
<br>
<br>
-
'''electroelution Purification'''<br>
 
-
PSB1C3 backbone
 
-
 
==='''2011.09.14-24'''===
==='''2011.09.14-24'''===
-
 
-
'''PCR'''<br><br>[[File:Cats2.gif |right|404px|caption]]
 
-
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
+
'''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
Line 267: Line 274:
<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
Line 307: Line 319:
<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
Line 319: Line 334:
<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
Line 333: Line 347:
'''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
    caption
  • 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
caption



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]
caption



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