Team:TzuChiU Formosa/Notebook/photopaper

From 2011.igem.org

(Difference between revisions)
(2011.09.10-11)
(Photopaper)
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[[File:Catt.gif|right|250px|caption]]
[[File:Catt.gif|right|250px|caption]]
'''Rhodobacter rudrum medium'''
'''Rhodobacter rudrum medium'''
-
<br>K2HPO4            1g
+
<br>
-
<br>NaCl             0.5g
+
<br>K2HPO4              1g
-
<br>FeSO4.7H2O         0.01g
+
<br>NaCl                0.5g
-
<br>CaCl2             0.02g
+
<br>FeSO4.7H2O            0.01g
-
<br>MnCl2.4H2O         0.002g
+
<br>CaCl2               0.02g
-
<br>MgSO4.7H2O         0.2g
+
<br>MnCl2.4H2O           0.002g
-
<br>NaMO2O4.2H2O        0.01g
+
<br>MgSO4.7H2O            0.2g
 +
<br>NaMO2O4.2H2O          0.01g
<br>ddH2O             998.258 ml
<br>ddH2O             998.258 ml
<br>________________________________________
<br>________________________________________
Line 141: Line 142:
          + 
          + 
-
<br>Yeast Extrat              0.5g
+
<br>Yeast Extrat                     0.5g
<br>Sodium malate
<br>Sodium malate
<br>(Sodium succinate dibasic hexohydrate)  5g
<br>(Sodium succinate dibasic hexohydrate)  5g
-
<br>NH4Cl                 1g
+
<br>NH4Cl                          1g
-
<br>ddH2O                 893.5ml
+
<br>ddH2O                       893.5ml
<br>_________________________________________________
<br>_________________________________________________
-
<br>                    1L
+
<br>                            1L
-
 
+
<br>
<br>
<br>
'''Raise E. coli(PSB1C3)'''
'''Raise E. coli(PSB1C3)'''
Line 157: Line 158:
<br>
<br>
<br>
<br>
-
 
-
 
==='''2011.09.08-13'''===
==='''2011.09.08-13'''===
<br>
<br>
Line 167: Line 166:
'''Raise Rhodobacter rubrum'''
'''Raise Rhodobacter rubrum'''
<br>
<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>
==='''2011.09.09'''===
==='''2011.09.09'''===
'''Raise Gluconacetobacter hansenii'''
'''Raise Gluconacetobacter hansenii'''
 +
<br>
<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>
-
 
+
-
 
+
==='''2011.09.10-11'''===
==='''2011.09.10-11'''===
-
 
'''Digestion check of DNA'''
'''Digestion check of DNA'''
 +
<br>
<br>[PSB1C3/EcoRI]
<br>[PSB1C3/EcoRI]
<br>
<br>
-
DNA           500ng<br>
+
DNA            500ng<br>
-
10×buffer       5μl<br>
+
10×buffer           5μl<br>
-
BSA           5μl<br>
+
BSA             5μl<br>
-
EcoRⅠ          1μl<br>
+
EcoRⅠ            1μl<br>
-
ddH2O         29μl<br>
+
ddH2O           29μl<br>
_______________________________
_______________________________
<br>
<br>
-
total          50μl
+
total           50μl<br>
<br>
<br>
<br>
<br>
<br>
<br>
-
[PSB1C3/pstⅠ]
+
[pSB1C3/PstⅠ]
<br>
<br>
-
DNA           500ng<br>
+
DNA             500ng<br>
-
10×buffer          5μl<br>
+
10×buffer           5μl<br>
-
BSA             5μl<br>
+
BSA               5μl<br>
-
pstⅠ             1μl<br>
+
pstⅠ              1μl<br>
-
ddH2O           29μl<br>                                               
+
ddH2O             29μl<br>                                               
_________________________________
_________________________________
<br>
<br>
-
total            50μl
+
total             50μl<br>
-
 
+
<br>
-
[PSB1C3/EcoRⅠ+pstⅠ]
+
[PSB1C3/EcoRⅠ+PstⅠ]
<br>
<br>
-
DNA              500ng<br>
+
DNA             500ng<br>
-
10×buffer            5μl<br>
+
10×buffer           5μl<br>
-
BSA             5μl<br>
+
BSA               5μl<br>
-
EcoRⅠ            1μl<br>
+
EcoRⅠ              1μl<br>
-
pstⅠ             1μl<br>
+
pstⅠ              1μl<br>
ddH2O            28μl<br>               
ddH2O            28μl<br>               
__________________________________
__________________________________

Revision as of 14:11, 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             998.258 ml
________________________________________
                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