Team:NCTU Formosa/Notebook

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JUDGING FORM
NCTU_Formosa: Lab Note
Dry Lab

Week 3.13~3.19

Searching for more information about antifungal diseases

Searching for useful databases: CAMP, PhyAMP

Week 3.20~3.26

Started building relationship between host, pathogen, disease and peptides

Week 3.27~4.2

View more bioformatic tools: BLAST, COBLAT, CD search

Week 4.3~4.9

Searching for more information or paper about fungal diseases

Web crawler of Phi-base, Phytopath,PhytAMP,CAMP

Week 4.10~4.16

Started building our own database, the building method was established

Week 4.17~4.23

With the idea of BLAST, COBALT, CDsearch, began to group up different kinds of peptides

Week 5.1~5.6

Finished grouping up of peptides in new database

Started on studying the Scoring Card Methods (SCM)

Week 5.15~5.21

Had a meeting with Prof. HO, HSING-YING to learn more about

SCM and confirm previous concept.

Debug of program and database

Week 5.22~5.28

Web crawler of Uniport

Successfully established our primary SCM

Week 6.25~7.1

Ask government agencies for disease information

Disease information searching on the Internet

Week 7.2~7.8

Web crawler of weather information of Central Weather Bureau

Week 7.9~7.15

Arrange the information of weather

Arrange the information of diseases

Started organizing the website of Parabase

Week 7.16~7.22

Combining and analyzing data of diseases and weather condition

Week 7.23~7.29

Combining and analyzing data of diseases and weather condition

Week 7.30~8.5

Paused for the 5th iGEM Asia-Pacific Conference

Start to discuss the whole concept and the key vision of project and wiki pages

Week 8.6~8.12

Started to build the model of IoT forecast system

Week 8.13~8.19

Building the model of IoT forecast system with CNN

Week 8.20~8.26

IoT forecast system model adjust

Database data updated and utilized

Week 8.27~9.2

Tried to use other method to build and utilize the model (composite combination model)

Primary test for Parabase on server

Week 9.3~9.9

Tried to use other methods to build and utilize the model (composite combination model)

Week 9.10~9.16

Started to link to IoTtalk, testing sensors

Week 9.17~9.23

Ran the whole process and debug of IoT forecast system

Week 9.24~9.30

Started beautifying the website of Parabase

Week 10.01~10.07

Parabase online

Working more and harder on wiki pages

Still debug and utilize the whole system, including SCM model, Parabase and Iot forecast system

Wiki Freeze

Wet Lab

Week 6.18~6.24

Received and resuspended gBlock Sa-AFP1 basic part

Received and resuspended gBlock Sa-AFP2 basic part

Received and resuspended gBlock Rs-AFP2 basic part

Received and resuspended gBlock MiAMP1 basic part

Received and resuspended gBlock LaLTP basic part

gBlock amplification of Sa-AFP1 basic part (Pfu PCR)

gBlock amplification of Sa-AFP2 basic part (Pfu PCR)

gBlock amplification of Rs-AFP2 basic part (Pfu PCR)

gBlock amplification of MiAMP1 basic part (Pfu PCR)

gBlock amplification of LaLTP basic part (Pfu PCR)

Electrophoresis of PCR product

Digestion of PCR product

Ligation of digestion product

Transformation of Sa-AFP1 basic part ligation product

Transformation of Sa-AFP2 basic part ligation product

Transformation of Rs-AFP2 basic part ligation product

Transformation of MiAMP1 basic part ligation product

Transformation of LaLTP basic part ligation product

Week 6.25~7.1

Sequencing of MiAMP1- psB1A3 plasmid

Sequencing of Sa-AFP1- psB1A3 plasmid

Sequencing of Sa-AFP2- psB1A3 plasmid

Sequencing of Rs-AFP2- psB1A3 plasmid

Electrophoresis (check the biobrick from IGEM kit)

  • K1763424
  • K808003
  • K1763440
  • K1763438

Successful transformation of iGEM kit K1763424 to E. coil DH5 α

Successful transformation of iGEM kit K808003 to E. coil DH5 α

Successful transformation of iGEM kit K1763440 to E. coil DH5 α

Successful transformation of iGEM kit K1763438 to E. coil DH5 α

Week 7.2~7.8

Cultivation of transformed E. coil

Cultivation of E. coil Rosetta gami

Week 7.9~7.15

Sequencing of LaLTP - psB1A3 plasmid

Miniprep of pSB1A3 plasmid

Digestion of pSB1A3 plasmid

Electrophoresis for digestion

Cultivation of E. coil with pSB1A3 plasmid

Received and resuspended gBlock of predicted peptides

Week 7.16~7.22

Digestion of K808003 Cr plasmid

Digestion of K848008 Cr plasmid

Digestion of J23101+B0034 Kr plasmid

Ligation of K808003 Cr plasmid

Ligation of K848008 Cr plasmid

Ligation of J23101+B0034 Kr plasmid

Ligation of pSB1A3 plasmid

Start cloning of predicted peptides

gBlock amplification of predicted peptides

  • Brevinin-1E (B-1E)
  • Pelophylaxin-4 (Pe-4)
  • 1095.6 Da venom vasodilator peptide (1095.6)
  • Temporin-SN2 (T-SN2)

PfuPCR of predicted peptides sequences

  • Brevinin-1E (B-1E)
  • Pelophylaxin-4 (Pe-4)
  • 1095.6 Da venom vasodilator peptide (1095.6)
  • Temporin-SN2 (T-SN2)

Digestion of B-1E plasmid

Digestion of Pe-4 plasmid

Digestion of 1095.6 plasmid

Digestion of T-SN2 plasmid

Ligation of digestion product of B-1E

Ligation of digestion product of Pe-4

Ligation of digestion product of 1095.6

Ligation of digestion product of T-SN2

Ligation of digestion product of pSB1A3

Week 7.23~7.29

Transformation of B-1E ligation product

Transformation of Pe-4 ligation product

Transformation of 1095.6 ligation product

Transformation of T-SN2 ligation product

Week 7.30~8.5

Experiment paused for the 5th iGEM Asia-Pacific Conference

Week 8.6~8.12

Taq-PCR of E.coil with transformed gene B-1E

Taq-PCR of E.coil with transformed gene Pe-4

Cultivation of transformed E.coil with transformed gene B-1E

Cultivation of transformed E.coil with transformed gene Pe-4

Sonication E.coil Rosetta gami with pSB1A3

Transformation for both DH5 α & Rosatta-gami (RG)

  • MiAMP1-psB1A3
  • Sa-AFP1- psB1A3
  • Sa-AFP2- psB1A3
  • Rs-AFP2- psB1A3
  • LaLTP-psB1A3

Start cloning of predicted peptides

  • M-C
  • C-1.4

PfuPCR of predicted peptides sequences

  • M-C
  • C-1.4

Digestion of PCR product of M-C

Digestion of PCR product of C-1.4

Ligation of digestion product of M-C

Ligation of digestion product of C-1.4

Transformation of M-C ligation product

Transformation of C-1.4 ligation product

Week 8.13~8.19

Cultivation for more E.coil Rosatta-gami (RG)

Peptide tests for best efficiency produced by Rosatta-gami in different time

  • MiAMP1-psB1A3
  • Sa-AFP1- psB1A3
  • Sa-AFP2- psB1A3
  • Rs-AFP2- psB1A3
  • LaLTP-psB1A3

Miniprep of MiAMP1-psB1A3 plasmid

Miniprep of Sa-AFP1- psB1A3 plasmid

Miniprep of Sa-AFP2- psB1A3 plasmid

Miniprep of Rs-AFP2- psB1A3 plasmid

Miniprep of LaLTP-psB1A3 plasmid

Cultivation for testing logarithmic phase

  • MiAMP1 psB1A3
  • Sa-AFP1 psB1A3
  • Sa-AFP2 psB1A3
  • Rs-AFP2 psB1A3
  • LaLTP psB1A3
  • Rosatta-gami with Cr Kr

SDS-PAGE of produced protein check

  • MiAMP1 psB1A3 16HR
  • Sa-AFP1 psB1A3 16HR
  • Sa-AFP2 psB1A3 16HR
  • Rs-AFP2 psB1A3 16HR
  • LaLTP psB1A3 16HR
  • Rosatta-gami with CrKr 16HR

Optical Density test for testing logarithmic phase

  • MiAMP1 psB1A3 16HR 18HR 20HR 22HR
  • Sa-AFP1 psB1A3 16HR 18HR 20HR 22HR
  • Sa-AFP2 psB1A3 16HR 18HR 20HR 22HR
  • Rs-AFP2 psB1A3 16HR 18HR 20HR 22HR
  • LaLTP psB1A3 16HR 18HR 20HR 22HR

Cultivation for more plasmid of M-C

Cultivation for more plasmid of C-1.4

Miniprep of M-C plasmid

Miniprep of C-1.4 plasmid

Week 8.20~8.26

TaqPCR of M-C plasmid

TaqPCR of C-1.4 plasmid

Digestion of PCR product of M-C

Digestion of PCR product of C-1.4

Sequencing of Pe-4- pSB1A3 plasmid

Sequencing of B-1E- pSB1A3 plasmid

Cultivation for testing OD value

  • MiAMP1 psB1A3
  • Sa-AFP1 psB1A3
  • Sa-AFP2 psB1A3
  • Rs-AFP2 psB1A3
  • LaLTP psB1A3
  • Rosatta-gami with CrKr

Ligation of digestion product of M-C

Ligation of digestion product of C-1.4

Transformation of M-C ligation product

Transformation of C-1.4 ligation product

Week 8.27~9.2

SDS-PAGE of produced protein check

  • MiAMP1 psB1A3 16HR
  • Sa-AFP1 psB1A3 16HR
  • Sa-AFP2 psB1A3 16HR
  • Rs-AFP2 psB1A3 16HR
  • LaLTP psB1A3 16HR
  • Rosatta-gami with CrKr

Cultivation for peptides

  • MiAMP1 psB1A3
  • Sa-AFP1 psB1A3
  • Sa-AFP2 psB1A3
  • Rs-AFP2 psB1A3
  • LaLTP psB1A3
  • Rosatta-gami with CrKr

SDS-PAGE with urea added to make peptides refolded

  • MiAMP1 psB1A3
  • Sa-AFP1 psB1A3
  • Sa-AFP2 psB1A3
  • Rs-AFP2 psB1A3
  • LaLTP psB1A3
  • psB1A3
  • Rosatta-gami (normal)

Week 9.3~9.9

Cultivation for testing logarithmic phase (Rosatta-gami)

  • MiAMP1 psB1A3
  • Sa-AFP1 psB1A3
  • LaLTP psB1A3
  • Pe-4 psB1A3
  • B-1E psB1A3

Cultivation for plasmid

  • M-C
  • C-1.4

Miniprep for sequencing plasmid

  • M-C
  • C-1.4

Week 9.10~9.16

Sequencing of M-C plasmid sample

Repeat DNA experiments for validated peptides, MiAMP1, Sa-AFP1, Sa- AFP2, Rs-AFP2, LaLTP

Pfu-PCR for MiAMP1, Sa-AFP1, Sa-AFP2, Rs-AFP2, LaLTP Digestion for PCR product

Ligation for MiAMP1, Sa-AFP1, Sa-AFP2, Rs-AFP2, LaLTP plasmids

Transformation for ligation product

Sonication for peptide

  • MiAMP1
  • LaLTP
  • Sa-AFP1
  • Sa-AFP2
  • Rs-AFP2
  • Pe-4
  • B-1E

Week 9.17~9.23

SDS-PAGE with urea added to make peptides refolded

  • MiAMP1
  • LaLTP
  • Sa-AFP1
  • Sa-AFP2
  • Rs-AFP2
  • Pe-4
  • B-1E

Week 9.24~9.30

Miniprep of Rs-AFP2, LaLTP plasmid for sequencing

Transformation for more E.coil to produce more plasmid

Wiki Freeze

Fungal Experiment

Week 4.24~4.30

First visit to Dr. Tsai at Taiwan Agricultural Research Institute, Taichung

First visit to Professor Lee, Department of Plant Pathology of National Chung Hsing University

Got some experimental fungi from Taiwan Agricultural Research Institute

Week 5.1~5.7

Second visit to Dr. Tsai.

Week 5.8~5.14

Started wet lab fungal experiments design

Started fungi culture (plates from Dr. Tsai)

Week 7.16~7.22

Started wet lab fungal experiment training for more team members

Started training experiments

Week 8.13~8.19

Visit Professor Chia-Lin Chung, Department of Plant Pathology and Microbiology, National Taiwan University

Week 8.27~9.2

Experiments failed, debug and adjusted experiment methods and protocols

Week 9.3~9.9

Visited Dr. Tsai again

Week 9.10~9.16

Adjusted and tested new method

Week 9.17~9.23

Inhibition zone test using NaN3 against Botrytis cinerea (training)

Spore germination test using NaN3 against Botrytis cinerea (training)

Plant infection test of Botrytis cinerea

Week 9.24~9.30

Spore germination test using sonication product of Sa-AFP1 transformed E. coli liquid against Botrytis cinerea

Spore germination test using sonication product of Sa-AFP2 transformed E. coli liquid against Botrytis cinerea

Spore germination test using sonication product of Rs-AFP2 transformed E. coli liquid against Botrytis cinerea

Plant infection test of Botrytis cinerea

Week 10.01~10.07

Spore germination test using sonication product of Sa-AFP1 transformed E. coli liquid against Botrytis cinerea

Spore germination test using sonication product of Sa-AFP1 transformed E. coli liquid against Botrytis cinerea

Spore germination test using sonication product of Sa-AFP2 transformed E. coli liquid against Botrytis cinerea

Spore germination test using sonication product of Rs-AFP2 transformed E. coli liquid against Botrytis cinerea

Plant infection test of Botrytis cinerea

Week 10.08~10.14

Inhibition zone test using Pe-4 pure peptide against Botrytis cinerea

Inhibition zone test using M-C pure peptide against Botrytis cinerea

Inhibition zone test using 1095.6 pure peptide against Botrytis cinerea

Inhibition zone test using T-SN2 pure peptide against Botrytis cinerea

Inhibition zone test using B-1E pure peptide against Botrytis cinerea

Inhibition zone test using C-1,4 pure peptide against Botrytis cinerea

Spore germination test using Pe-4 pure peptide against Botrytis cinerea

Spore germination test using M-C pure peptide against Botrytis cinerea

Spore germination test using 1095.6 pure peptide against Botrytis cinerea

Spore germination test using T-SN2 pure peptide against Botrytis cinerea

Spore germination test using B-1E pure peptide against Botrytis cinerea

Spore germination test using C-1,4 pure peptide against Botrytis cinerea

Plant infection test using Pe-4 pure peptide against Botrytis cinerea

Plant infection test using M-C pure peptide against Botrytis cinerea

Plant infection test using 1095.6 pure peptide against Botrytis cinerea

Plant infection test using T-SN2 pure peptide against Botrytis cinerea

Plant infection test using B-1E pure peptide against Botrytis cinerea

Plant infection test using C-1,4 pure peptide against Botrytis cinerea

Plant infection test using Pe-4 pure peptide against Botrytis cinerea

Plant infection test using M-C pure peptide against Botrytis cinerea

Plant infection test using 1095.6 pure peptide against Botrytis cinerea

Plant infection test using T-SN2 pure peptide against Botrytis cinerea

Plant infection test using B-1E pure peptide against Botrytis cinerea

Plant infection test using C-1,4 pure peptide against Botrytis cinerea

Week 10.15~10.21

Result double confirm of Inhibition zone test

  • Pe-4
  • M-C
  • 1095.6
  • T-SN2
  • B-1E
  • C-1,4

Result double confirm of Spore germination test

  • Pe-4
  • M-C
  • 1095.6
  • T-SN2
  • B-1E
  • C-1,4

Result double confirm of Plant infection test

  • Pe-4
  • M-C
  • 1095.6
  • T-SN2
  • B-1E
  • C-1,4

Week 10.21~10.28

Result triple confirm of Inhibition zone test

  • Pe-4
  • M-C
  • 1095.6
  • T-SN2
  • B-1E
  • C-1,4

Result triple confirm of Spore germination test

  • Pe-4
  • M-C
  • 1095.6
  • T-SN2
  • B-1E
  • C-1,4

Result triple confirm of Plant infection test

  • Pe-4
  • M-C
  • 1095.6
  • T-SN2
  • B-1E
  • C-1,4

Week 10.29~11.04

Spore germination test of validated peptides

Inhibition zone test of validated peptides

Result confirmed and checked

Wiki Freeze

Untitled Document