Team:TzuChiU Formosa/Notebook/photopaper

From 2011.igem.org

(Difference between revisions)
(2011.09.27)
Line 142: Line 142:
-
<font size=4 color="#000080">Protocols</font>
+
<font size=4 color="#000080"><b>Protocols</b></font>
<Hr Align="left" width="100%" size=3>
<Hr Align="left" width="100%" size=3>
<font size=3>2011.09.07</font>
<font size=3>2011.09.07</font>
<br>[[File:Catt.gif|right|250px|caption]]
<br>[[File:Catt.gif|right|250px|caption]]
-
'''Rhodobacter rudrum medium'''
+
Rhodobacter rudrum medium
<br>
<br>
-
<br>K2HPO4                   1g<br>
+
<br>K2HPO4               1g<br>
-
<br>NaCl                   0.5g<br>
+
<br>NaCl              0.5g<br>
-
<br>FeSO4.7H2O             0.01g<br>
+
<br>FeSO4.7H2O             0.01g<br>
-
<br>CaCl2                0.02g<br>
+
<br>CaCl2              0.02g<br>
-
<br>MnCl2.4H2O              0.002g<br>
+
<br>MnCl2.4H2O           0.002g<br>
-
<br>MgSO4.7H2O                        0.2g<br>
+
<br>MgSO4.7H2O           0.2g<br>
-
<br>NaMO2O4.2H2O                       0.01g<br>
+
<br>NaMO2O4.2H2O           0.01g<br>
-
<br>ddH2O             998.258ml<br>
+
<br>ddH2O              998.258ml<br>
<br>________________________________________
<br>________________________________________
-
<br>                1L →take100ml<br>
+
<br>                 1L →take100ml<br>
          + 
          + 
-
<br>Yeast Extrat                              0.5g<br>
+
<br>Yeast Extrat                          0.5g<br>
<br>Sodium malate
<br>Sodium malate
<br>(Sodium succinate dibasic hexohydrate)    5g<br>
<br>(Sodium succinate dibasic hexohydrate)    5g<br>
Line 171: Line 171:
<br>
<br>
<br>
<br>
-
'''Raise E. coli(PSB1C3)'''
+
<br><font color="#000000" size=2><b>Raise E. coli(PSB1C3)</B></font>
<br>
<br>
<br>1.50ml LB+500μl CHLORAMPHENICOL
<br>1.50ml LB+500μl CHLORAMPHENICOL

Revision as of 04:41, 4 October 2011

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)


Contents

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


Digestion of DNA

[pSB1C3/EcoRⅠ+PstⅠ]

DNA             500ng

10×buffer           5μl

BSA              5μl

EcoRⅠ             1μl

pstⅠ              1μl

ddH2O            28μl

__________________________________

total            50μl

→37℃ for 30 mins


[pSB1C3/XbaⅠ+SpeⅠ]

DNA               10μl

10×buffer            5μl

BSA               5μl

XbaⅠ               1μl

SpeⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs


[pSB1C3/SpeⅠ+PstⅠ]

DNA               10μl

10×buffer            5μl

BSA               5μl

SpeⅠ              1μl

pstⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs


[pSB1C3/EcoRⅠ+XbaⅠ]

DNA               10μl

10×buffer            5μl

BSA               5μl

EcoRⅠ              1μl

XbaⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs


[pSB1A3/EcoRⅠ+PstⅠ]

DNA               10μl

10×buffer            5μl

BSA               5μl

EcoRⅠ              1μl

PstⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs

[pSB1A3/EcoRⅠ+SpeⅠ]

DNA               10μl

10×buffer            5μl

BSA               5μl

EcoRⅠ              1μl

SpeⅠ               1μl

ddH2O              28μl

____________________________________

total              50μl

→37℃ for 2 hrs

electroelution Purification

[pSB1C3/EcoRⅠ+PstⅠ]

[pSB1C3/XbaⅠ+SpeⅠ]

[pSB1C3/SpeⅠ+PstⅠ]

[pSB1C3/EcoRⅠ+XbaⅠ]

[pSB1A3/EcoRⅠ+PstⅠ]

[pSB1A3/EcoRⅠ+SpeⅠ]


2011.09.12-20


PCR


[acsAB]

[acsCD]

[CCcax-Ccp]

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



electroelution Purification

[acsAB]

[acsCD]

[CMCax-Ccp]


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


[acsCD/XbaⅠ+SpeⅠ]

DNA            10μl

10×buffer         5μl

BSA             5μl

XbaⅠ            1μl

SpeⅠ             1μl

ddH2O            28μl

__________________________________
total               50μl

→37℃ for 16 hr


[CMCax/SpeⅠ+AlwNⅠ]

DNA            10μl

10×buffer         5μl

BSA             5μl

SpeⅠ            1μl

AlwNⅠ             1μl

ddH2O            28μl

__________________________________

total               50μl

→37℃ for 16 hr


[Ccp/AlwNⅠ+PstⅠ]

DNA            10μl

10×buffer         5μl

BSA             5μl

AlwNⅠ            1μl

PstⅠ             1μl

ddH2O            28μl

__________________________________
total               50μl

→37℃ for 16 hr


electroelution Purification

[acsAB/ XbaⅠ+SpeⅠ]

[acsCD/XbaⅠ+SpeⅠ]

[CMCax/SpeⅠ+AlwNⅠ]

[Ccp/AlwNⅠ+PstⅠ]


2011.09.22


Ligation of DNA


[pSB1C3-acsAB]
caption



Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


[pSB1A3-acsCD]

Vector           3μl

Insert           14μl

ligase buffer       2μl

ligase            1μl

ddH2O             -μl

_________________________________

total           20μl

→16℃ for 16 hr


[pSB1C3-acsCD]

Vector           3μl

Insert           14μl

ligase buffer       2μl

ligase            1μl

ddH2O             -μl

_________________________________

total           20μl

→16℃ for 16 hr


[pSB1C3-CMCax-Ccp]

Vector           3μl

Insert           14μl

ligase buffer       2μl

ligase            1μl

ddH2O             -μl

_________________________________

total           20μl

→16℃ for 16 hr


2011.09.23


PCR

[R0011 promoter]

R0011 promoter        1μl

5×Buffer            4μl

2.5μM dNTP          1.6μl

Taq               0.2μl

ddH2O             13.2μl

_____________________________________

total              20μl


electroelution Purification

[R0011 promoter]

Transformation of DNA

[pSB1C3-acsAB]

Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr


[pSB1C3-acsCD]

Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr

[pSB1A3-acsCD]

Transform into E.coli
LB+Ampicillin
→37℃ for 14 hr


[pSB1C3-CMCax-Ccp]

Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr


Digestion of DNA

[R0011 promoter/EcoRⅠ+XbaⅠ]

DNA           10μl

10×buffer        5μl

BSA           5μl

EcoRⅠ          1μl

XbaⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr

electroelution Purification

[R0011 promoter/EcoRⅠ+XbaⅠ]


2011.09.24


Ligation of DNA

[pSB1C3-R0011]

Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


[R0011-acsAB]

Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr

[R0011-acsCD]

Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


2011.09.24


Digestion of DNA

[R0011-acsAB/EcoRⅠ+SpeⅠ]

DNA           10μl

10×buffer        5μl

BSA           5μl

EcoRⅠ          1μl

SpeⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr

[R0011-acsCD/EcoRⅠ+SpeⅠ]

DNA           10μl

10×buffer        5μl

BSA           5μl

EcoRⅠ          1μl

SpeⅠ           1μl

ddH2O          28μl

____________________________

total             50μl

→37℃ for 16 hr


electroelution Purification

[R0011-acsAB/EcoRⅠ+SpeⅠ]

[R0011-acsCD/EcoRⅠ+SpeⅠ]


2011.09.25


Ligation of DNA

[pSB1C3-R0011-acsAB]

Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr


[pSB1C3-R0011-acsCD]

Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr

2011.09.26


Transformation of DNA

[pSB1C3-R0011-acsAB]

Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr


[pSB1C3-R0011-acsCD]

Transform into E.coli
LB+CHLORAMPHENICOL
→37℃ for 14 hr

Digestion of DNA

[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]

DNA           10μl

10×buffer        5μl

BSA           5μl

SpeⅠ          1μl

PstⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr

electroelution Purification

[pSB1C3-R0011-acsCD/SpeⅠ+PstⅠ]


2011.09.27


Ligation of DNA

[pSB1C3-R0011-acsCD-CMCax-Ccp]

Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr

Digestion of DNA

[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]

DNA           10μl

10×buffer        5μl

BSA           5μl

PstⅠ          1μl

EcoRⅠ           1μl

ddH2O          28μl

____________________________


total             50μl

→37℃ for 16 hr

electroelution Purification

[pSB1C3-R0011-acsCD-CMCax-Ccp/PstⅠ+EcoRⅠ]


2011.09.28


Ligation of DNA

[pSB1A3-R0011-acsCD-CMCax-Ccp]

Vector          3μl

Insert          14μl

ligase buffer       2μl

ligase          1μl

ddH2O           -μl

________________________________

total          20μl

→16℃ for 16 hr

2011.09.29


Transformation of DNA

[pSB1C3-R0011-acsAB]
[pSB1A3-R0011-acsAB-CMCax-Ccp]

Transform into E.coli
LB+Ampiclin+CHLORAMPHENICOL
→37℃ for 14 hr