HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

Notebook

Week 1: Jul. 31th - Aug. 6th

No experiments were conducted.

We had been planning the project of this year.

Week 2: Aug. 7th - Aug. 13th

Sunday
- Transformation of BBa_R0040(2011 distribution 1-6I)
Monday
- Cultivation of E.coli which has the complex we made last year for the next day's mini-prep.
- Cultivation of E.coli which has been transformed yesterday for the next day's mini-prep.
Tuesday
- Replacing Arabinose promoter (BBa_I0500) of the complex by TetR promoter (BBa_R0040)
  - mini-prep:
  - PCR (EX_RBS_SlrP_F / PS_SlrP_R) → gel extraction. This Parts will be used as insert.
Wednesday
- Promoter replacing (continuation from yesterday)
  - digestion
    - Insert (RBS-SlrP-GFP-double terminator): XbaI, PstI
    - Vector (TetR promoter which is on pSB1A2 vector): SpeI, PstI
  - Gel extraction and Ethanol precipitation of digestion products.
  - Ligation and transformation(in DH5-alpha).
Thursday
- Promoter replacing (continuation from Tuesday)
  - Result of yesterday's transformation: FAILIRE because of over cultivation.
- Re-try
  - Insert PCR and Gel extraction.
  - Digestion, Gel extraction, and Ethanol precipitation.
  - Ligation, Transformation, and Cultivation.
Friday
- Promoter replacing (Re-try, continuation from yesterday)
  - Result of yesterday's transformation: FAILURE because of mistake of ligation
- Promoter replacing (Re-Re-try)
  - Insert PCR and Gel extraction.
Saturday
- Promoter replacing (Re-Re-try, continuation from yesterday)
  - digestion, Gel extraction and Ethanol precipitation.
  - Ligation, transformation, and Cultivation.

Week 3: Aug. 14th - Aug. 20th

Sunday
- promoter Replacing (Re-Re-try)
  - Result: SUCCEED
  - Single colony isolation of the E.coli and cultivate it in LBA (liquid)
Monday
- Backbone Construction
  - We find irrelevant BsaI site in pSB1A2 Vector, so we decide that replace vector pSB1A2 to pSB1K3
  - mini-prep of cultivated E.coli
    - FAILURE: wrong protocol. DNA maight be shone
  - Single colony isolation (again)
- Verification of Promoter Replacing - 1
  - idea: We use 2010 complex as substrate, and this is on pSB1T3 Vector. However, after replasing, the product is on pSB1A2 vector because TetR Promoter is on pSB1A2. So, cultivating the product in LBT, and then confirm that E.coli in LBT does not increase.
  - cultivating E.coli in LBT.
Tuesday
- Verification of Plomoter Replacing
  - result: SUCCEED. E.coli in LBT did not decidedly increase.
- mini-prep of E.coli for backbone constructing
  - FAILURE: Cultivating E.coli in LB. We can't know what other kinds of Bacteria are in the medium.
- Verification of Plimoter Replacing -2
  - idea: taking some kinds of PCR, and checking the length of its products by electrophoresis.
    1. EX_F / PS_R: expected length=2.7kbp
    2. X_NLS*3_GFP / 200dn_PS_R: expected length=2kbp
    3. 100up_EX_F / PS_SlrP_R: expected length=1.3kbp
  - result: SUCCEED. We observed appropriate bands.
Wednesday
- Backbone constructing
  - mini-prep(again-again)
  - PCR of the complex with EX_F / PS_R.
    - The product (EX-TetR-RBS-SlrP-NLS-NLS-NLS-GFP-dt-TetR-RBS-RFP-dt-SP) will be insert.
  - Gel extraction of PCR product.
  - digestion
    - insert: EcoRI / PstI
    - vector(pSB1K3, distributed from iGEM HQ): EcoRI / PstI
  - Gel extraction
Thursday
- Backbone constructing
  - Ethanol precipitation of yesterday's digestion product.
  - Ligation, transformation, and cultivation.
Friday
- Backbone constructing
  - vector replacing
    - result: SUCCEED.
Saturday
- No experiments because required primers had not arrived.

Week 4: Aug. 21th - Aug. 27th

Sunday
- No experiments because required primers had not arrived.
Monday
- cultivate E.coli which has plasmid that its promoter and vector were replaced.(for next day's mini-prep)
Tuesday
- mini-prep of cultivated E.coli
Wednesday
- No experiments because required primers had not arrived.
Thursday
- preparation of next day's PCR
  - idea: The complex has two double terminators, so we were anxious that the PCR product would be full of smear because our forward primer is annealed to 5' terminal of double terminator seaquence. Thus, we thought it is nice to cut away the unwanted double terminator by digesting with endonucleases, NdeI and CpoI. The recognition site of NdeI is in GFP coding sequence, and the one of CpoI is in RFP sequence.
    - digestion of the comples with NdeI / CpoI (Both of them were stocked in our laboratory.)
      - result: FAILURE. We think the endonucleases are too old.
Friday
- Primers came.
- Backbone constructing
  - final PCR
    - Because of yesterday's failure of double terminator removing, we must deal with the primer mis-binding problem. So we decided to adopt "Step-Down PCR" protocol.
    - PCR: BsaI_SlrP_R / BsaI_dt_F Extension time is 2-minutes (because KOD_Plus_Neo will extent 1kbp/30sec, the manual says and PCR product will be 3.0kbp)
    - result: FAILURE. Extension time might be too short.
Saturday
- Backbone construction
  - final PCR (Re-try)
    - We set the extension time 4 minutes
    - result: SUCCEED. We obserbed appropriate band by electrophoresis
  - Gel extraction of PCR product

Week 5: Aug. 28th - Sep. 3rd

Sunday
- No experiment
Monday
- No experiment
- Meeting: we report the completion of backbone, and discuss the plan of next step.
Tuesday
- No experiment
Wednesday
Thursday
- Making linear backbone plasmid: Inserting Fluorescent Protein coding sequence
  - Selecting insert DNA: CFP(BBa_E0020) and RFP(BBa_E1010)
  - Xba-byebye PCR of insert DNA
  - Gel extraction
  - Digestion
    - insert(Sba-byebye-PCRed): NotI / SpeI
    - vector(BsaI backbone): BsaI(2 cutting sites are there)
  - Gel extraction and Ethanol precipitation
  - Ligation, transformation, and cultivation.
Friday
- Making plasmid
  - result: FAILURE
  - Discussing the cause.
    - We decide re-make the backbone from the beginning.
      - single colony isolation and cultivation of E.coli which has last year complex
Saturday
- first step: promoter replacing (AraC → TetR)
  - mini-prep
  - PCR and gel extraction
  - Digestion
    - insert(EX_SlrP......RFP_dt_SP): XbaI / PstI
    - vector(pSB1A2 with TetR): SpeI / PstI
  - Gel extraction and Ethanol precipitation
  - Ligation, transformation, and cultivation.

Week 6: Sep. 4th - Sep. 10th

Sunday
- backbone construcion (Re-try)
  - yesterday's promoter replacing result: SUCCEED. We observed fluorescence of GFP.
- second step: backbone replacing (pSB1A2 → pSB1K3)
  - PCR and gel extraction of insert(EX-TetR-RBS......RFP-dt-SP)
  - Digestion
    - Insert: EcoRI / PstI
    - Vector: EcoRI / PstI
  - Gel extraction
Monday
- backbone construction (Re-try)
  - second step: vector replacing
    - Ethanol precipitation
    - Ligation, transformation, and cultivation
Tuesday
- backbone construction (Re-try)
  - second step: vector replacing
    - result: FAILURE. The transformed E.coli didn't increse on LBK plate.
Wednesday
- backbone construction (Re-try)
  - second step: vector replacing (Re-try)
Thursday
Friday
Saturday