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 which has STEP 1 product.
    - Cultivating 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 of the E.coli which has STEP 1 product.
    - Cultivating in liquid LBA.
- 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 of the E.coli which has STEP 1 product.
    - Cultivating in liquid LBA.
- Verification of Plimoter Replacing -part 2
  - IDEA: taking some kinds of PCR, and checking the length of its products by electrophoresis.
    - Template: STEP 1 product
    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
- Construction of BsaI backbone -1st try
  - STEP 2 :vector Replacing -3rd try
    - mini-prep of cultivated E.coli.
    - PCR of insert fragment.
      - Template: STEP 1 product
      - F primer: EX-F
      - R primer: PS_R
        Product: EX-TetR-RBS-SlrP-NLS-NLS-NLS-GFP-dt-TetR-RBS-RFP-dt-SP
    - Gel extraction of PCR product.
    - Digestion
      - insert: EcoRI, PstI
      - vector(pSB1K3, distributed from iGEM HQ): EcoRI, PstI
    - Gel extraction
Thursday
- Construction of BsaI backbone -1st try
  - STEP 2 :vector Replacing -3rd try
    - Ethanol precipitation of yesterday's digestion product.
    - Ligation.
    - Transformation.
    - Cultivation.
Friday
- Construction of BsaI backbone -1st try
  - STEP 2 :vector Replacing -3rd try
    - 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
- Construction of BsaI backbone -1st try
  - STEP 3: Adding BsaI cloning site -1st try
    - Cultivation of E.coli which has STEP 2 product for next day's mini-prep.
Tuesday
- Construction of BsaI backbone -1st try
  - STEP 3: Adding BsaI cloning site -1st try
    - Mini-prep of cultivated E.coli.
Wednesday
- No experiments because required primers had not arrived.
Thursday
- Construction of BsaI backbone -1st try
  - STEP 3: Adding BsaI cloning site -1st try
    - Preparation of next day's PCR
      - IDEA: PCR template DNA (STEP 2 product) 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 STEP 2 product: NdeI, CpoI
      - result: FAILURE. We think the endonucleases are too old.
Friday
- Primers came.
- Construction of BsaI backbone -1st try
  - STEP 3: Adding BsaI cloning site -1st try
    - PCR with primers which have tails of BsaI cloning site.
      - Templete: STEP 2 product
      - F primer: BsaI_dt_F
      - R primer: BsaI_SlrP_R
      - Extension time: 2-minutes (Because the manual says that KOD_Plus_Neo (polymerase) will extend 1kbp/30sec, and PCR product will be 3.0kbp)
      - Because we fail to remove double terminator, we must deal with the primer mis-binding problem. So we decided to adopt "Step-Down PCR" protocol.
    - result: FAILURE. Extension time might be too short.
Saturday
- Construction of BsaI backbone -1st try
  - STEP 3: Adding BsaI cloning site -2nd try
    - PCR (on equal recipe).
      - 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　(linear), and discuss the plan of next step.
Tuesday
- No experiment
Wednesday
- No experiment
Thursday
- Construction of BsaI backbone -1st try
  - STEP 4: Making circular plasmid -1st try: Inserting Fluorescent Protein coding sequence.
    - Selecting insert DNA: CFP(BBa_E0020) and RFP(BBa_E1010)
    - PCR of insert DNA
      - Templete: BBa_E0020 / BBa_E1010
      - F primer: Xba-byebye-prefix-F
      - R primer: 200bp_dn_R
    - Gel extraction
    - Digestion
      - insert(Xba-byebye-PCRed): NotI, SpeI
      - vector(BsaI backbone): BsaI(2 cutting sites are there)
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation.
Friday
- Construction of BsaI backbone -1st try
  - STEP 4: Making circular plasmid -1st try
  - result: FAILURE
    - Discussing the cause.
      - We decide re-make the backbone from the beginning.
        Because we couldn't draw a conclusion.
      - Single colony isolation and cultivation of E.coli which has 2010 complex
Saturday
- Construction of BsaI backbone -2nd try
  - STEP 1: Promoter replacing (AraC → TetR)
    - Mini-prep of cultivated E.coli.
    - 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.
    - Digestion
      - insert: XbaI, PstI
      - vector (pSB1A2 with TetR): SpeI, PstI
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation.

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

Sunday
- Construction of BsaI backbone -2nd try
  - STEP 1: Promoter replacing
    - Result: SUCCEED. We observed fluorescence of GFP.
  - STEP 2: Replacing vector (pSB1A2 → pSB1K3)
    - PCR of insert
      - Templete: STEP 1 product
      - F primer: EX_F
      - R primer: PS_R
        Product: EX-TetR-RBS-SlrP-NLS-NLS-NLS-GFP-dt-TetR-RBS-RFP-dt-SP
    - Digestion
      - Insert: EcoRI, PstI
      - Vector: EcoRI, PstI
    - Gel extraction
Monday
- Construction of BsaI backbone -2nd try
  - STEP 2: Replacing vector
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation on LBK plate.
Tuesday
- Construction of BsaI backbone -2nd try
  - STEP 2: Replacing vector
    - Result: FAILURE. The transformed E.coli didn't increse on LBK plate.
Wednesday
- Construction of BsaI backbone -2nd try
  - STEP 2: Replacing vector.
    - PCR of insert fragment (on equal recipe)
    - Gel extraction.
    - Digestion (on equal recipe).
    - Gel extraction.
Thursday
- Construction of BsaI backbone -2nd try
  - STEP 2: Replacing vector.
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation.
Friday
- Construction of BsaI backbone -2nd try
  - STEP 2: Replacing vector.
    - result: SUCCEED
Saturday
- No experiments.

Week 7: Sep. 11th - Sep. 17th

Sunday
- Construction of BsaI backbone -2nd try
  - STEP 3: Adding BsaI cloning site -1st try
    - PCR with primers which have tails of BsaI cloning site.
      - Templete: STEP 2 product
      - F primer: BsaI_dt_F
      - R primer: BsaI_SlrP_R
        SUCCEED. We observed an appropriate band.
    - Gel extraction.
Monday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -1st try: Inserting Fluorescent Protein coding sequence.
    - PCR of insert DNA
      - Templete: BBa_E0020 / BBa_E1010
      - F primer: Xba-byebye-prefix-F
      - R primer: 200bp_dn_R
    - Gel extraction
    - Digestion
      - insert(Xba-byebye-PCRed): NotI, SpeI
      - vector(BsaI backbone): BsaI(2 cutting sites are there)
    - Gel extraction.
Tuesday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -2nd try
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation on LBK plate.
Wednesday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -1st try
    - Result: FAILURE
Thursday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -2nd try
    - PCR of insert (on equal recipe).
    - Gel extraction.
    - Digestion (on equal recipe).
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivate on LBT plate.
Friday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -2nd try
    - result: FAILURE
      - We found a problem of SpeI/NotI double digestion. We thought that NotI would work with M buffer (ToYoBo or TaKaRa-Bio, same as NEBuffer4), but NotI will be work only in H Buffer (same as NEBuffer3), BSA and Triton must be added. So we cannot digest simultaneously. Thus, we decided to digest one by one.
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -3rd try
    - Digestion -1st stage (There were Xba-byebye-PCRed product left)
      - NotI (H Buffer with BSA and Triton) *There are two NotI sites in PCRed fragment.
    - Gel extraction.
    - Digestion -2nd stage
      - SpeI (M Buffer)
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation (There are digested and ethanol precipitated vector left)
    - Transformation.
    - Cultivation on LBK plate.
Saturday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -3rd try
    - result: FAILURE
      - Researching more on SpeI activity, we got to know that SpeI can't work on terminal of fragment. So, we have to digest with SpeI before NotI digestion.

Week 8: Sep. 18th - Sep. 24th

Sunday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -4th try
    - Xba-byebye-PCR (on equal recipe)
    - Gel extraction.
    - Digestion -stage 1
      - SpeI (M Buffer)
    - Gel extraction.
    - Digestion -stage 2
      - NotI (H Buffer with BSA and Triton) *One of the two NotI sites were cut away by SpeI digestion.
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation on LBK plate.
Monday
- Construction of BsaI backbone -2nd try
  - STEP 4: Making circular plasmid -4th try
    - result: FAILURE
    - To elucidate the cause, we took the sequence of the product.
      - primer: SlrP_373_to_394_F
        result: It is revealed that PCR in STEP 3 (adding BsaI cloning site) was unsuccessful. We thought the cause was "two dT problem (see Week4 Thursday)"
Tuesday
- Construction of BsaI backbone -3rd try
  - We change the process of making backbone entirely. We would construct the backbone from scratch.
  - STEP 1: Picking out RBS-SlrP part from 2010 complex
    - PCR
      - Templete: 2010 complex (There were mini-prep product left)
      - F primer: EX_RBS_SlrP_F
      - R primer: PS_SlrP_R
        Product: EX_RBS_SlrP_SP
    - Gel extraction.
  - STEP 2: Making TetR-RBS-SlrP part
    - PCR product (STEP 1) is used as insert. Vector is pSB1A2 which has TetR Biobrick.
    - Digestion
      - Insert: XbaI, PstI
      - Vector: SpeI, PstI
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation on LBA plate.
Wednesday
- Construction of BsaI backbone -3rd try
  - STEP 3: Making the templete plasmid of STEP 4 (Adding BsaI site)
    - Single colony isolation * 30colony from LBA plate.
    - Colony PCR of each bacterial suspension.
      - Selecting colonies which STEP 2 was successful.
    - Cultivation of selected E.coli in liquid LBA.
Thursday
- Construction of BsaI backbone -3rd try
  - STEP 3: Making the templete plasmid of STEP 4 (Adding BsaI site)
    - 3-piece ligation
      - Vector: pSB1K3
      - Insert1: STEP 2 product
      - Insert2: double Terminator Biobrick (BBa_B0015)
    - Mini-prep of cultivated E.coli.
    - PCR of inserts.
      - Templete: STEP 2 product
      - F primer: EX_F
      - R primer: PS_R
        Product: EX-TetR-RBS-SlrP-SP
      - Templete: BBa_B0015
      - F primer: EX_F
      - R primer: PS_R
        Product: EX-dt-SP
    - Gel extraction.
    - Digestion
      - Insert1: EcoRI, SpeI
      - Insert2: XbaI, PstI
      - Vector: EcoRI, PstI
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation.
    - Cultivation on LBK plate.
Friday
- Construction of BsaI backbone -3rd try
  - STEP 4: Adding BsaI site
    - Single colony isolation * 30colony from LBK plate.
    - Colony PCR of each bacterial suspension.
      - Selecting colonies which STEP 3 was successful.
    - Cultivation of selected E.coli in liquid LBK.
Saturday
- Construction of BsaI backbone -3rd try
  - STEP 4: Adding BsaI site
    - PCR
      - Templete: STEP 3 product
      - F primer: BsaI_dt_F
      - R primer: BsaI_SlrP_R
        Product: BsaI backbone (linear DNA fragment. Insert must be ligated.)
    - Gel extraction.

Week 9: Sep. 25th - Oct. 1st

Sunday
- Construction of BsaI backbone -3rd try
  - STEP 5: Making circular plasmid -1st try
    - Xba-byebye-PCR of insert fragments.
      - Templete: BBa_E0040 and BBa_E1010
      - F primer: Xba_byebye_prefix_F
      - R primer: 200dn_PS_R
    - Gel extraction.
    - Digestion -1st stage
      - Inserts: SpeI
      - Vector: BsaI
    - Gel extraction.
    - Digestion -2nd stage
      - Inserts: NotI
    - Gel extraction.
    - Ethanol precipitation.
    - Ligation.
    - Transformation in K12 heat-shocked competent cell (K12 has T3SS gene)
    - Cultivation on LBK plate.
Monday
- Construction of BsaI backbone -3rd try
  - STEP 5: Making circular plasmid -1st try
    - result: SUCCEED. We observed fluorescence.
- Preparation for part registration.
  - Single colony isolation from LBK plate.
  - Cultivate in liquid LBK.
Tuesday
- NEW PROJECT: GSK Tag System (See here)
  - SUMMARY: As we wrote in abstract, we want to screen domains to see which ones can be injected by T3SS system. So, we need a simple system with which we can see whether the domain was injected or not. Using this system, we can judge the injectability with antibody. It is comparatively easy.
  - Order a primer (BsaI_GSK_SlrP_R).
- Preparation for part registration.
  - Mini-prep of cultivated E.coli.
  - Sequencing.
    - Primer 1: 100up_EX_F
    - Primer 2: SlrP_373_to_394_F
    - Primer 3: PS_R
    - Primer 4: 200dn_PS_R
      - result: We were able to sequence full length (pSB1K3-EX-TetR-RBS-SlrP-BsaIsite-GFP-BsaIsite-dt-SP-pSB1K3). Although a mutation in SlrP coding sequence was found, it was silent. No problems had occurred.
  - Purification by gel extraction (extract with TE).
  - FAILURE. The concentration was too low, it was under 25ng/ul.
- Preparation for part registration -Re-challenge
  - Single colony isolation.
  - Cultivate in liquid LBK.
Wednesday
- Preparation for part registration
  - Mini-prep of cultivated E.coli.
  - Purification.
  - Testing its concentration.
    - result: about 40ng/ul. OK.
Thursday
- GSK Tag System
  - Processing insert fragment.
    - We choose 8 different domains to inject. (In fact, we wanted to screen more large number of domains, but there were no time.)
    - Xba-byebye-PCR
      - Temolete: See here
      - F primer: Xba_byebye_prefix_F
      - R primer: 200dn_PS_R
    - Gel extraction.
    - Digestion -1st step
      - SpeI
    - Gel extraction.
    - Digestion -2nd step
      - NotI
    - Gel extraction.
Friday
- GSK Tag System
  - Construction of BsaI backbone with GSK Tag
    - Primer has come.
    - PCR to add GSK Tag and BsaI cloning sites.
      - Templete: Product made on Sep. 22th
      - F primer: BsaI_dt_F
      - R primer: BsaI_GSK_SlrP_R
      - Step-Down protocol, extention time is 4 minutes.
    - Gel extracion.
    - Digestion
      - Vector: BsaI
    - Gel extraction.
    - Ethanol precipitation (Vector and inserts)
    - Ligation.
    - Transformation.
    - Cultivation on LBK plate.
Saturday
- Selecting of yesterday's products
  - Colony PCR. 10 sample per insert.