Team:TzuChiU Formosa/Notebook/photopaper

From 2011.igem.org

(Difference between revisions)
(Protocols)
(Protocols)
Line 129: Line 129:
'''Rhodobacter rudrum medium'''
'''Rhodobacter rudrum medium'''
<br>
<br>
-
<br>K2HPO4                  1g
+
<br>K2HPO4                  1g<br>
-
<br>NaCl                   0.5g
+
<br>NaCl                   0.5g<br>
-
<br>FeSO4.7H2O            0.01g
+
<br>FeSO4.7H2O            0.01g<br>
-
<br>CaCl2                0.02g
+
<br>CaCl2                0.02g<br>
-
<br>MnCl2.4H2O              0.002g
+
<br>MnCl2.4H2O              0.002g<br>
-
<br>MgSO4.7H2O                        0.2g
+
<br>MgSO4.7H2O                        0.2g<br>
-
<br>NaMO2O4.2H2O                        0.01g
+
<br>NaMO2O4.2H2O                        0.01g<br>
-
<br>ddH2O             998.258ml
+
<br>ddH2O             998.258ml<br>
<br>________________________________________
<br>________________________________________
-
<br>                1L →take100ml
+
<br>                1L →take100ml<br>
          + 
          + 
-
<br>Yeast Extrat                             0.5g
+
<br>Yeast Extrat                             0.5g<br>
<br>Sodium malate
<br>Sodium malate
-
<br>(Sodium succinate dibasic hexohydrate)    5g
+
<br>(Sodium succinate dibasic hexohydrate)    5g<br>
-
<br>NH4Cl                          1g
+
<br>NH4Cl                          1g<br>
-
<br>ddH2O                         893.5ml
+
<br>ddH2O                         893.5ml<br>
<br>_________________________________________________
<br>_________________________________________________
-
<br>                            1L
+
<br>                            1L<br>
<br>
<br>
<br>
<br>
Line 247: Line 247:
'''PCR'''
'''PCR'''
<br>[[File:Cats2.gif |right|404px|caption]]
<br>[[File:Cats2.gif |right|404px|caption]]
-
<br>template DNA   1μl
+
<br>template DNA   1μl<br>
-
<br>5×Buffer     4μl
+
<br>5×Buffer     4μl<br>
-
<br>2.5μM dNTP    1.6μl
+
<br>2.5μM dNTP    1.6μl<br>
-
<br>10μM F      1μl
+
<br>10μM F      1μl<br>
-
<br>10μM R      1μl
+
<br>10μM R      1μl<br>
-
<br>Taq        0.2μl
+
<br>Taq        0.2μl<br>
-
<br>ddH2O      8.8μl
+
<br>ddH2O      8.8μl<br>
<br>_______________________________
<br>_______________________________
<br>
<br>
-
<br>total       20μl
+
<br>total       20μl<br>
<br>
<br>
<br>
<br>
Line 266: Line 266:
<br>[acsAB/ XbaⅠ+SpeⅠ]
<br>[acsAB/ XbaⅠ+SpeⅠ]
<br>
<br>
-
<br>DNA           10μl
+
<br>DNA           10μl<br>
-
<br>10×buffer        5μl
+
<br>10×buffer        5μl<br>
-
<br>BSA           5μl
+
<br>BSA           5μl<br>
-
<br>EcoRⅠ          1μl
+
<br>EcoRⅠ          1μl<br>
-
<br>pstⅠ           1μl
+
<br>pstⅠ           1μl<br>
-
<br>ddH2O          28μl
+
<br>ddH2O          28μl<br>
<br>____________________________
<br>____________________________
<br>
<br>
-
<br>total             50μl
+
<br>total             50μl<br>
<br>→37℃ for 16 hr
<br>→37℃ for 16 hr
<br>
<br>
Line 281: Line 281:
<br>[acsCD/XbaⅠ+SpeⅠ]
<br>[acsCD/XbaⅠ+SpeⅠ]
<br>
<br>
-
<br>DNA            10μl
+
<br>DNA            10μl<br>
-
<br>10×buffer         5μl
+
<br>10×buffer         5μl<br>
-
<br>BSA             5μl
+
<br>BSA             5μl<br>
-
<br>EcoRⅠ            1μl
+
<br>EcoRⅠ            1μl<br>
-
<br>pstⅠ             1μl
+
<br>pstⅠ             1μl<br>
-
<br>ddH2O            28μl
+
<br>ddH2O            28μl<br>
<br>__________________________________
<br>__________________________________
<br>
<br>
-
total               50μl
+
total               50μl<br>
<br>→37℃ for 16 hr
<br>→37℃ for 16 hr
<br>
<br>
Line 298: Line 298:
<br>[PSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]]
<br>[PSB1C3-acsAB][[File:Cats3.jpg|right|404px|caption]]
<br>
<br>
-
<br>Vector          3μl
+
<br>Vector          3μl<br>
-
<br>Insert          14μl
+
<br>Insert          14μl<br>
-
<br>ligase buffer      2μl
+
<br>ligase buffer      2μl<br>
-
<br>ligase          1μl
+
<br>ligase          1μl<br>
-
<br>ddH2O           -μl
+
<br>ddH2O           -μl<br>
<br>________________________________
<br>________________________________
<br>
<br>
-
<br>total          20μl
+
<br>total          20μl<br>
<br>
<br>
<br>
<br>
Line 312: Line 312:
<br>[PSB1A3-acsCD]
<br>[PSB1A3-acsCD]
<br>
<br>
-
<br>Vector           3μl
+
<br>Vector           3μl<br>
-
<br>Insert           14μl
+
<br>Insert           14μl<br>
-
<br>ligase buffer       2μl
+
<br>ligase buffer       2μl<br>
-
<br>ligase            1μl
+
<br>ligase            1μl<br>
-
<br>ddH2O             -μl
+
<br>ddH2O             -μl<br>
<br>_________________________________
<br>_________________________________
<br>
<br>
-
<br>total           20μl
+
<br>total           20μl<br>
<br>
<br>
<br>
<br>
Line 327: Line 327:
'''PCR'''
'''PCR'''
<br>
<br>
-
<br>PR0011 promoter       1μl
+
<br>PR0011 promoter       1μl<br>
-
<br>5×Buffer            4μl
+
<br>5×Buffer            4μl<br>
-
<br>2.5μM dNTP          1.6μl
+
<br>2.5μM dNTP          1.6μl<br>
-
<br>Taq               0.2μl
+
<br>Taq               0.2μl<br>
-
<br>ddH2O              13.2μl
+
<br>ddH2O              13.2μl<br>
<br>_____________________________________
<br>_____________________________________
<br>
<br>
-
<br>total              20μl
+
<br>total              20μl<br>
<br>
<br>
<br>
<br>
Line 366: Line 366:
<br>[PSB1C3-promoter]
<br>[PSB1C3-promoter]
<br>
<br>
-
<br>Vector            3μl
+
<br>Vector            3μl<br>
-
<br>Insert            14μl
+
<br>Insert            14μl<br>
-
<br>ligase buffer        2μl
+
<br>ligase buffer        2μl<br>
-
<br>ligase            1μl
+
<br>ligase            1μl<br>
-
<br>ddH2O             -μl
+
<br>ddH2O             -μl<br>
<br>________________________________
<br>________________________________
-
<br>total             20μl
+
<br>total             20μl<br>
<br>
<br>

Revision as of 15:16, 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