Team:TzuChiU Formosa/Notebook/photopaper

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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

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
                                                  (+
______________________________________________________
                                        50μl
→37℃ for 16 hr

'''Digestion of DNA'''
acsCD/ XbaⅠ+SpeⅠ
DNA					10μl
10×buffer				5μl
BSA					5μl
EcoRⅠ					1μl
pstⅠ					1μl
ddH2O					28μl
                                                  (+
______________________________________________________
                                        50μl
→37℃ for 16 hr


2011.09.22

'''Ligation of DNA'''
PSB1C3-acsAB
Vector                                  3μl
Insert                                  14μl
ligase buffer                           2μl
ligase                                  1μl
ddH2O                                   -μl
                                                  (+
______________________________________________________
                                        20μl

'''Ligation of DNA'''
PSB1A3-acsCD
Vector                                  3μl
Insert                                  14μl
ligase buffer                           2μl
ligase                                  1μl
ddH2O                                   -μl
                                                 (+
______________________________________________________
                                        20μl


2011.09.23

'''PCR'''
PR0011 promoter                         1μl
5×Buffer				4μl
2.5μM dNTP				1.6μl
Taq		                        0.2μl
ddH2O					13.2μl
                                                  (+
______________________________________________________
                                        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
                                                  (+
______________________________________________________
                                        20μl