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
- Construction of BsaI backbone -1st try
  - STEP 1 : Replacing Arabinose promoter by TetR promoter -1st try
    - Transformation of TetR part (BBa_R0040, 2011 distribution 1-6I).
    - Cultivation of transformed E.coli on LBA plate.
Monday
- Construction of BsaI backbone -1st try
  - STEP 1 : Replacing Arabinose promoter by TetR promoter -1st try
    - Cultivation of glycerol stocked E.coli which has 2010 product in liquid LBT for the next day's mini-prep.
    - Single colony isolation from LBT plate, and cultivation of E.coli which has TetR part in liquid LBA for the next day's mini-prep.
Tuesday
- Construction of BsaI backbone -1st try
  - STEP 1: Promoter Replacing -1st try: To replace AraC promoter of GFP by TetR promoter.
    - Mini-prep of cultivated E.colis.
    - PCR of insert fragment.
      - Templete: 2010 complex
      - F primer: EX_RBS_SlrP_F
      - R primer: PS_SlrP_R
        Product: EX-RBS-SlrP-NLS-NLS-NLS-GFP-dT-TetR-RBS-RFP-dT-SP
    - Gel extraction pf PCR product.
Wednesday
- Construction of BsaI backbone -1st try
  - STEP 1 :Promoter Replacing -1st try
    - Digestion
      - Insert (PCR product): XbaI, PstI
      - Vector (pSB1A2 vector with TetR): SpeI, PstI
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation (in DH5-alpha).
    - Cultivation on LBA plate.
Thursday
- Construction of BsaI backbone -1st try
  - STEP 1 :Promoter Replacing -1st try
    - Result: FAILIRE, because of over cultivation.
  - STEP 1 :Promoter Replacing -2nd try
    - PCR of insert fragment (on equal recipe).
    - Gel extraction.
    - Digestion (on equal recipe)
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation (in DH5-alpha).
    - Cultivation on LBA plate.
Friday
- Construction of BsaI backbone -1st try
  - STEP 1 :Promoter Replacing -2nd try
    - Result: FAILURE, because of mistake of ligation.
  - STEP 1 :Promoter Replacing -3rd try
    - PCR of insert fragment (on equal recipe).
    - Gel extraction.
Saturday
- Construction of BsaI backbone -1st try
  - STEP 1 :Promoter Replacing -3rd try
    - digestion (on equal recipe).
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - transformation (in DH5-alpha).
    - Cultivation on LBA plate.

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

Sunday
- Construction of BsaI backbone -1st try
  - STEP 1 :Promoter Replacing -3rd try
    - Result: SUCCEED
- Construction of BsaI backbone -1st try
  - STEP 2 :vector Replacing -1st try
    - We find irrelevant BsaI site in pSB1A2 Vector, so we decide that replace vector pSB1A2 to pSB1K3
    - Single colony isolation of the E.coli.
    - Cultivate it in liquid LBA.
Monday
- Construction of BsaI backbone -1st try
  - STEP 2 :vector Replacing -1st try
    - mini-prep of cultivated E.coli.
    - FAILURE, wrong protocol. DNA maight be shone.
  - STEP 2 :vector Replacing -2nd try
    - Single colony isolation.
- Verification of Promoter Replacing -part 1
  - Cultivating E.coli in LBT.
    - 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.
Tuesday
- Verification of Plomoter Replacing - part 1
  - result: SUCCEED, E.coli in LBT did not decidedly increase.
- Construction of BsaI backbone -1st try
  - STEP 2 :vector Replacing -1st try
    - mini-prep of cultivated E.coli.
    - FAILURE, because of cultivating E.coli in liquid LB by mistake. Other unwanted kinds of Bacteria might be in the medium.
  - STEP 2 :vector Replacing -3rd try
    - Single colony isolation.
- Verification of Plimoter Replacing -part 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)
    - PCR, gel extraction
    - digestion
      - insert: EcoRI. PstI
      - vector: EcoRI, PstI
    - gel extraction
Thursday
- backbone construction (Re-try)
  - second step: vector replacing (Re-try)
    - Ethanol precipitation
    - Ligation, transformation, and cultivation
Friday
- backbone construction (Re-try)
  - second step: vector replacing (Re-try)
    - result: SUCCEED
Saturday
- No experiments.