Team:TzuChiU Formosa/Notebook/photopaper

From 2011.igem.org

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(2011.09.24)
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'''Ligation of DNA'''
'''Ligation of DNA'''
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<br>[pSB1C3-promoter]
<br>[pSB1C3-promoter]
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Revision as of 17:41, 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


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]

[cmcax-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


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        1μl

5×Buffer            4μl

2.5μM dNTP          1.6μl

Taq               0.2μl

ddH2O             13.2μl

_____________________________________

total              20μl


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


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

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