Team:Paris Bettencourt/Notebook/Odor Library

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Notebook

June

June 26th
Goal

Design plasmid for limonene synthase

Procedure

Used MG1665 strain, the RBS with the highest initial rate and the sequence of r-limonene synthase from genBank. Added polyhistidine tail to the construct. Submitted the construct to Jake.

Results

Designed plasmid for limonene synthase using the software geneious. Found a biobricks part containing this sequence. For the purpose of saving budget, we would first transform this standard part into cell and later modify it.

June 27th
Goal

Transform the bioBricks limonene synthase(BBa_I742111) part into e.coli.
We found that the bioBricks kit for 2014 contain the limonene synthase sequence in plate 4 position 3I.

Procedure

  1. Start thawing the competent cells(Used Christina's competent cells) on ice.
  2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
  3. Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
  4. Close tubes and incubate the cells on ice for 30 minutes.
  5. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
  6. Incubate the cells on ice for 5 minutes.
  7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
  8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
  9. For the control, label two petri dishes with LB agar (CM). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
  10. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

Results

Transformed the plasmid containing limonene synthase sequence from biobricks into E.Coli. Colonies seen on both plates. Glycerol Stock made: PB. 018 and PB. 019

June 28th
Goal

Made chemical competent cell following standard protocol with MG1665 strains.

Procedure

  1. The night before, inoculate a 5 ml culture and grow overnight with selection.
  2. The day of the experiment dilute cells ~ 1:200 into selective media. For this example add 250 ul to 50 ml of selective media. Note: The protocol is easily scaled to increase the number of cells.
  3. Grow the cells to an OD600 of 0.6 – 0.7. Use a large flask, 500ml, for good aeration. Use a baffled flask for fastest growth. This takes about 3 hours depending on the cells. Medium-heavy cloudiness by eye is fine.
  4. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes. Note: Keep the cells at 4 ºC from now on.
  5. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2. Leave on ice 4 hours to overnight.
  6. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
  7. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
  8. Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.

Note: Frozen cells are only good once.Do not refreeze cells once thawed.

Results

The transformed cell were grown for 20 hours and the transformation was successful. The plates were put into the 4 degree fridge for making stocks on Monday.


July

July 1st
Goal

To design the primers and gBlock to modify the BioBricks part BBa_I742111.

Procedure

  1. decide the major steps of modification
  2. to PCR the biobricks construct using primers that have Agel restriction site sequences.
  3. to digest the PCR product
  4. to PCR the gBlock with Promoter, RBS and restriction sites (Xbal and Agel)
  5. to digest the PCR product
  6. to ligate the two sequences together
  7. 2. Design the primers for PCR and the gBlock including promoter, RBS and restriction sites using the software Geneious.

Results

  • fwd primer for BioBricks part PCR: 0PB.015
  • rev primer for BioBricks: 0PB.016
  • gBlock with promoter, RBS and restriction sites: 0PB. 019
  • fwd primer for gBlock PCR: 0PB.30
  • rev primer for gBlock PCR: 0PB.31
  • (We ended up ordering the gBlock as synthetic gene sequence, because it is too long for oligos and too short for gBlocks. Oh well.)

July 7th
Goal

to extract plasmid of biobricks I742111 from the transformed cells.

Procedure

  1. Growth the cell for two days
  2. Centrifuge the tube for 15 minutes with 4000 rps
  3. Follow the standard protocol of Thermo Scientific miniprep kit
  4. Label the centrifuge tube and store it in the -20 freezer. (Syl miniprep)

Results

  1. We grew two tubes of glycerol stocks. However living cells were only seen in one of them, which was used for miniprep.
  • Plasmid stored in the box after the procedure. PCR would be done tomorrow.

    • July 8th
    Goal

    Amplify limonene synthase gene sequence for E.coli

    • Fw primer: oPB.015
    • Rv primer: oPB.016

    Procedure

    ReagentVolume
    1x
    Nuclease-free water71 ul
    5x Phusion HF Buffer20 ul
    10 mM dNTPs2 ul
    Forward Primer (10 uM)1 ul
    Reverse Primer (10 uM)1 ul
    Template Plasmid1 ul
    Phusion DNA Polymerase1 ul
    DMSO3 ul
    Total Volume100 ul
    TemperatureTimeFunction
    start98 C30 secmelt
    cycle 198 C10 secmelt
    cycle 251 C30 secanneal
    cycle 372 C30 secextend
    finish72 C5 minextend
    blind10 Cforeverblind

    Results

    PCR product at the expected length.

    July 9th
    Goal

    1. To run the Gel of the PCR product to confirm that the segment is at the right length.
    2. to digest the segment using AgeI and XbaI. Purify the digested product.

    Procedure

      • Take the PCR tubes out of the machine.
      • Mixing the dye with PCR products, from each tube.
      • Run the gel for an hour and half using 100bp plus DNA ruler
      • Observe under UV light
      • Digest in the 37 degree incubator for 10 minutes using AgeI and XbaI.
        • ReagentVolume
        Nuclease-free water27.5 ul
        10x FD Buffer5 ul
        DNA12.5 ul
        AgeI2.5 ul
        Xbal2.5 ul
        Total Volume50 ul
      • Purify following the protocol of digestion purification kit. Used warm NF water for diluting DNA at the last step.

    Results

    The PCR product is at expected length. Purified product has a very low concentration(2.5ng/ul), meaning the PCR product was a mixture of multiple segments and didn't give ideal result. Therefore proceed to PCR again.

    July 16th
    Goal

    Amplify limonene synthase gene sequence for E.coli

    • Fw primer: oPB.015
    • Rv primer: oPB.016

    Procedure

    ReagentVolume
    1x
    Nuclease-free water71 ul
    5x Phusion HF Buffer20 ul
    10 mM dNTPs2 ul
    Forward Primer (10 uM)1 ul
    Reverse Primer (10 uM)1 ul
    Template Plasmid1 ul
    Phusion DNA Polymerase1 ul
    DMSO3 ul
    Total Volume100 ul
    TemperatureTimeFunction
    start98 C30 secmelt
    cycle 198 C10 secmelt
    cycle 251 C30 secanneal
    cycle 372 C2.5 minextend
    finish72 C5 minextend
    blind10 Cforeverblind

    Results

    It worked!

    Goal

    : transform the PB19 synthase part into e.coli.

    Procedure

    1. Start thawing the competent cells(Used Christina's competent cells) on ice.
    2. Add 50 µL of thawed competent cells into pre-chilled 2ml tube, and another 50µL into a 2ml tube, labelled for your control.
    3. Add 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Make sure to keep the competent cells on ice.
    4. Close tubes and incubate the cells on ice for 30 minutes.
    5. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
    6. Incubate the cells on ice for 5 minutes.
    7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmid backbones with antibiotic resistance other than ampicillin.
    8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid backbone, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
    9. For the control, label two petri dishes with LB agar (CM). Plate 20 µl and 200 µl of the transformation onto the dishes, and spread.
    10. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

    Results

    Colonies seen on both plates.

    July 17th
    Goal

    to extract plasmid of PB19 from the transformed cells.

    Procedure

    1. Growth the cell overnight
    2. Centrifuge the tube for 15 minutes with 4000 rps
    3. Follow the standard protocol of Thermo Scientific miniprep kit
    4. Label the centrifuge tube and store it in the -20 freezer. (Syl mini prep)

    Results

    • Tube 1: 40 ng/ul
    • Tube 2: 30ng/ul

    Goal

    to digest the segment using AgeI and XbaI. Purify the digested product.

    Procedure

    Digest in the 37 degree incubator for 10 minutes using AgeI and XbaI.

    ReagentVolume
    Nuclease-free water27.5 ul
    10x FD Buffer5 ul
    DNA12.5 ul
    AgeI2.5 ul
    Xbal2.5 ul
    Total Volume50 ul

    Purify following the protocol of digestion purification kit. Used warm NF water for diluting DNA at the last step

    Results

    Concentration after purification: 12.2 ng/ul

    July 23rd
    Goal

    : to achieve the great great goal of magnifying the RBS and promoter before digestion, purification and ligation.

    Procedure

    we did a nano drop of the mini prep plasmid, and it is 460ng/ul. The curve looks good. We diluted 1:500

    Results

    a fainted band was seen. However, the concentration after purification is below 5 ng/ul, too low to be used to ligation. Therefore, we decided to design the sequence as two oligos, with Xbal and Agel sticky ends.


    August

    August 4th
    Goal

    to dilute received oilgos, to Phosphorylate them, to Anneal them and to ligation is with Limonene vector

    Procedure

    1. Dilute the oilgo to 100 uM, the stock
    2. Take 10 ul out of the 100 uM stock and dilute to make 10 uM working stock
    3. Mix:
      • 2 uL 10 µM sense oligo
      • 2 uL 10 µM anti-sense oligo
      • 2 uL 10 x PNK (polynucleotide kinase) buffer A
      • 2 uL 10mM ATP
      • 1 uL T4 polynucleotide kinase (PNK)
      • 10 uL distilled water
      • to give 20 uL total volume
    4. Incubate at 37C for 30 mins
    5. Place in boiling water bath for 2 min, then remove water bath from the heat source and allow the reaction (still in the water bath) to cool to room temperature. (Labelled Annealing in the box)
    6. ligation
    7. Mix :
      • 8 ul of vector (12 ng/ul)
      • 1 ul of insert
      • 0.5 ul of ligase enzyme
      • 2 ul of ligase buffer
      • 8.5 ul of distilled water
      • total 20 ul
      • put under 22 degree for 30 minutes
      • put under 16 degree for 30 minutes
      • put in 4 degree overnight

    Results

    colonies were seen after transforming and plating.

    August 10th
    Goal

    to sequence the ligated plasmid for limonene construct

    Procedure

    • Dilute the mini prep product 1: 2 (Give us concentration 70ng/ul and 80ng/ul)
    • design primers that are about 200 bp away upstream and downstream from the inserted sequence
    • Send them to GATC

    Results

    It appears that the old plasmid recirculate itself.

    Further Steps

    regrow 6 more colonies, minipreped and did analytical PCR with NotI and AgeI Colony 1a appears to have the complete plasmid.

    August 20th
    Goal

    to PCR ilvBN gene from MG1655

    Procedure

    1. Picked 2 colonies from the plate MG1655 115+GFP
    2. Inoculate them each in 50 ul of NF H2O
    3. Boiled for 3 mins at 98 degree
    4. Use the solution as DNA template
    5. Follow standard PCR protocol from this step on
    ReagentVolume
    1x
    Nuclease-free water63 ul
    5x Phusion HF Buffer20 ul
    10 mM dNTPs2 ul
    Forward Primer (10 uM)5 ul
    Reverse Primer (10 uM)5 ul
    Template Plasmid1 ul
    Phusion DNA Polymerase1 ul
    DMSO3 ul
    Total Volume100 ul

    TemperatureTimeFunction
    start98 C30 secmelt
    cycle 198 C10 secmelt
    cycle 251 C30 secanneal
    cycle 372 C2 minextend
    finish72 C5 minextend
    blind10 Cforeverblind

    Results

    Band seen on both sample (from 2 colonies)

    August 21st
    Goal

    to Digest the PCR product (ilvBN) from yesterday and to digest pET-DUET1 as vector

    Procedure

    Vector (pET-DUET1)

    ReagentVolume
    Nuclease-free water38 ul
    10x FD Buffer5 ul
    DNA 2 ul
    EcoRI2 ul
    NcoI2 ul
    FastAP1 ul
    Total Volume50 ul

    ilvBN

    ReagentVolume
    Nuclease-free water39 ul
    10x FD Buffer5 ul
    DNA 2 ul
    EcoRI2 ul
    NcoI2 ul
    Total Volume50 ul

    Incubate at 37 degree for 2 hours. Purification using the PCR purification kit.

    Results

    24 ng/ul for C2 and 12 ng/ul for C1 (Will use C2 PCR purified product for digestion.)

    August 25th
    Goal

    to verify the insert of RBS and promoter is in the limonene biobrick part.

    Procedure

    digest

    ReagentVolume
    Nuclease-free water19 ul
    10x FD Buffer3 ul
    DNA1 ul
    AgeI1 ul
    Xbal1 ul
    Total Volume25 ul

    (Miniprep concentration: 500 for N.1, 400 for N.2 and N.3) Expected length:
    3750 and 3740
    or 3840 and 3740

    Results

    (with 100bp plus DNA ladder)

    (the control sample didn’t get digested. Therefore, we can’t say for sure if the ligation worked. However, we decided to go ahead with sequencing and see the result.)

    August 26th
    Goal

    To ligate ilvBN gene with PETDUET 1

    Procedure
    ReagentVolume
    Nuclease-free water 3 ul
    Ligase Buffer2 ul
    ligase 1 ul
    Insert(2132 bp, 10ng/ul)4 ul
    Vector(5381bp, 18ng.ul)10 ul
    Total Volume20 ul

    25 degree for 1 hour and then heat shock transformation.

    Results

    colonies seen. Picked 4 colonies for analytical digestion. Bands seen at expected length.

    August 27th
    Goal

    to ligate the aldB gblock with pSB1C3

    Procedure

    Digested and Purified PCR product, concentration: 40 ng/ul
    Digested and purified vector pSB1C3: 20 ng/ul

    ReagentVolume
    Nuclease-free water 10 ul
    Ligase Buffer2 ul
    ligase 1 ul
    Insert(2132 bp, 10ng/ul)4 ul
    Vector(5381bp, 18ng.ul)4.5 ul
    Total Volume20 ul

    20 degree for 5 hours, followed by heat shock transformation into NEB strain.

    Results

    Blurring band seen on gel after colony PCR, will process to mini prep and sequencing

    5/06 Odourless coli

    11/06 Odourless coli
    tnaA deletion mutant from Keio Collection
    Location: 63:E:9

    Streaked on LBA+Kan
    We have glycerol stock sPB.000

    5/06 Banana and Wintergreen E. coli Transformation

    Goal:
    Transform NEB Turbo E. coli competent cells (à la Jake) using standard heat shock transformation protocol (à la Jake).
    The generators do not include promoters, therefore there is no expression of the odour generating enzymes.
    I will transform the following parts:
    • pPB.003 BBa_R0040 2013 3 5E pSB1C3
    • pPB.004 BBa_J45199 2013 4 6I pSB1C3
    • pPB.005 BBa_J45119 2012 2 5B pSB1AT3
    Procedure:
    1. Resuspend DNA from registry plates using 10 uL of ddH2O. Make sure to resuspend GOOD.
    2. Follow protocol below. Using 1 uL of DNA.
    Protocol: Heat Shock Transformation of E. coli 1) Place 20 ul of cells in a pre-chilled Eppendorf tube.
    2) Add 1 ul of DNA to the chilled cells
    3) Mix gently by flicking the tube.
    4) Chill on ice for 10 minutes.
    5) Heat shock at 42 °C for 30 seconds.
    6) Return to ice for 2 minutes.
    7) Add 200 ul LB medium and recover the cells by shaking at 37 °C.Another rich medium can substitute for the recovery.
    The recovery time varies with the antibiotic selection.
    Tetracycline: 60-120 minutes
    Chloramphenicol: 60-120 minutes
    8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate.
    Use glass beads to spread the cells.

    6/06 Transformations

    Yesterday's plates do not have colonies.

    ToDo:
    • Plate the 200 uL from yesterday's tube containing recovered cells.
      • pPB.003 BBa_R0040 LBA+Cm
      • pPB.004 BBa_J45199 LBA+Cm
      • pPB.005 BBa_J45119 LBA+Tet
    • Transform:
      • pPB.003 BBa_R0040: Constitutive TetR promoter
        • Resuspended DNA from 2013:3:5E
        • pSB1C3 Chloramphenicol resistance
      • pPB.004 BBa_J45199: Banana generator without promoter
        • Resuspended DNA from 2013:4:6I
        • pSB1C3 Chloramphenicol resistance
        • Requires promoter and 5mM isoamyl alcohol
      • pPB.005 BBa_J45119: Wintergreen generator without promoter
        • Resuspended DNA from 2012:2:5B
        • pSB1AT3 Ampicillin and Tetracycline resistance
        • Requires promoter and 2 mM Salicylic acid
      • USED, NOT AVAILABLE BBa_J45200: Constitutive Banana generator
        • 2012:2:5F
        • pSB1AT3 Ampicillin and Tetracycline resistance
        • Requires 5mM isoamyl alcohol
      • USED, NOT AVAILABLE BBa_J45120: Constitutive Wintergreen generator
        • 2012:2:5D
        • pSB1AT3 Ampicillin and Tetracycline resistance
        • Requires 2 mM Salicylic acid
    Protocol: Heat Shock Transformation of E. coli
    • Plate fresh transformations:
      • pPB.003 BBa_R0040 LBA+Cm
      • BBa_J45119 LBA+Tet
      • BBa_J45199 LBA+Cm
    • Streak NEB Turbo in LBA for singles (tomorrow set an overnight for competent cell production).

    7/06 Single colonies from Transformations
    Took single colonies, started overnight cultures.

    8/06 Single colonies from Transformations


    Left the overnight cultures… will need to dilute for mini prepping.


    9/06 Single colonies from Transformations


    No growth. Picked single colonies and started overnights again.

    10/06 Minipreps

    Minipreps:

    pPB.003: BBa_R0040
    pPB.004: BBa_J45199
    pPB.005 BBa_J45119

    All samples are at high concentrations ~100 ng/uL

    26/06 Digestion set up

    Digestion:

    FD Restriction Digestion
    Reagent Volume (uL)
    NF ddH2O
    17
    Buffer
    3
    FD Enzyme I
    1
    FD Enzyme II
    1
    DNA
    3
    Total Volume
    25


    Cut pPB.003 with SpeI & PstI
    Expected fragments:
    1. 2.1 kb
    2. 18 bp
    Cut pPB.004 with XbaI and PstI
    Expected fragments:
    1. ~ 2 kb
    2. 1.765 kb
    Cut pPB.005 with XbaI and PstI
    Expected fragments:
    1. 3.4 kb
    2. 1.26 kb
    I performed a purification with the PCR cleanup kit and stored the samples at -20ºC.

    27/06 SAGE set up


    Sage:
    SAGE on thick gel (3 mm) 1% agarose should be fine.
    There should be two bands for pPB.004 and pPB.005.

    Gel extraction:
    1. Cut out the second band of pPB.004 and pPB.005 (1.8 kb and 1.3 kb respectively).
    2. Weigh the agarose fragments (100 mg gel : 300 uL QG Buffer)
      • Max amount is 400 mg
    3. Solubilize agarose completely by incubating at 50ºC for 10 min in water bath. Vortexing the tube every 3 minutes helps.
    4. Color of mixture should be yellow. Otherwise add 10 uL of 3 M sodium acetate pH 5.
    5. Add the equivalent of 1 gel volume of isopronanol and mix the tube (although for fragments between 500 bp and 4 kb this should matter).
    6. Place column in a 2 mL collection tube
    7. Load sample into column. Centrifuge 1 min @ 13 000 rpm.
    8. Discard flowthrough and repeat adding 0.5 mL of QG buffer.
    9. Discard flowthorugh and wash with 0.75 mL of PE Buffer. Let the column sit for 5 min prior centrifugation.
    10. Discard flowthrough and centrifuge again to ensure all residual ethanol is gone.
    11. Place the column into a clean 1.5 mL Epp and elute by adding 50 uL of NF-ddH2O. Make sure to wait 2 min before centrifugation.
    12. Store at 4ºC overnight or -20ºC for longer periods.

    27/06 Ligation set up

    Ligation:

    July

    7/07 We start again...


    Digestion:

    FD Restriction Digestion
    Reagent Volume (uL)
    NF ddH2O
    17
    Buffer
    3
    FD Enzyme I
    1
    FD Enzyme II
    1
    DNA
    3
    Total Volume
    25
    Cut pPB.003 with SpeI & PstI
    Expected fragments:
    1. 2.1 kb
    2. 18 bp
    Cut pPB.004 with XbaI and PstI
    Expected fragments:
    1. ~ 2 kb
    2. 1.765 kb
    Cut pPB.005 with XbaI and PstI
    Expected fragments:
    1. 3.4 kb
    2. 1.26 kb
    SAGE:
    SAGE on thick gel (3 mm) 1% agarose should be fine.
    There should be two bands for pPB.004 and pPB.005.

    WEIRD GEL: Very dim bands after staining for 7 minutes in EtBr. The gel was ran at 100 mV but it took more than 60 minutes for the indicator marker dye to reach half migration. FAILED.

    14/07 Digestion-product gel extraction and ligation

    Use the remaining purified digestion products. Run a gel and purify the appropriate bands.


    Cut pPB.003 with SpeI & PstI
    Expected fragments:
    1. 2.1 kb
    2. 18 bp
    Cut pPB.004 with XbaI and PstI
    Expected fragments:
    1. ~ 2 kb
    2. 1.765 kb
    Cut pPB.005 with XbaI and PstI
    Expected fragments:
    1. 3.4 kb
    2. 1.26 kb
    Gel was good. The gel extraction not. Things got messy with the blade and the transilluminator, I chopped off half of the wrong band.

    17/07/2014 Restriction Digestion set up

    Digestion:

    FD Restriction Digestion
    Reagent Volume (uL)
    NF ddH2O
    75
    Buffer
    10
    FD Enzyme I
    2.5
    FD Enzyme II
    2.5
    DNA
    10
    Total Volume
    100

    Incubate at 37ºC with moderate shaking for ~1 hour.

    Cut pPB.003 with SpeI & PstI
    Expected fragments:
    1. 2.1 kb
    2. 18 bp
    Cut pPB.004 with XbaI and PstI
    Expected fragments:
    1. ~ 2 kb
    2. 1.765 kb
    Cut pPB.005 with XbaI and PstI
    Expected fragments:
    1. 3.4 kb
    2. 1.26 kb
    Cut pPB.014 with SpeI and PstI
    (For enhanced Banana pPB.016 and Wintergreen pPB.015)
    Expected fragments:
    1. ~2.1 kb
    2. ~18 kb

    21/07/2014 Ligation set up
    Ligation:
    1. Fermentas T4 DNA Ligation
      Reagent
      Vector (pPB.003)
      		 25 ng
      Insert
      		 75 ng
      T4 DNA Ligase Buffer 10X
      		 1 uL
      T4 DNA Ligase
      		0.5 uL
      NF-ddH2O
      		 as needed
      Total
      		 10 uL

      Fermentas T4 DNA Ligation
      Reagent
      Vector
      		 2 uL
      Insert
      		 2 uL
      NF ddH2O
      		 4.5 uL
      T4 DNA Ligase Buffer
      		 1 uL
      T4 DNA Ligase
      		 0.5 uL
      Total
      		 10 uL

    2. Incubate @22ºC for 10 minutes.
    3. Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
    No colonies. Failed experiment.

    pPB.016 Banana Generator under Anderson Promoter
    • pPB.016: Banana generator AndersonPromoter (BBa_J23108)
      • Requires 5mM isoamyl alcohol

    7/07 Transformations

    I will transform the following parts:
    • pPB.014 BBa_J23108 2014 4 4C pSB1C3
    1. Resuspend DNA from registry plates using 10 uL of ddH2O. Make sure to resuspend GOOD.
    2. Follow protocol below. Using 1 uL of DNA.
    Protocol: Heat Shock Transformation of E. coli
    1) Place 20 ul of cells in a pre-chilled Eppendorf tube.
    2) Add 1 ul of DNA to the chilled cells
    3) Mix gently by flicking the tube.
    4) Chill on ice for 10 minutes.
    5) Heat shock at 42 °C for 30 seconds.
    6) Return to ice for 2 minutes.
    7) Add 200 ul LB medium and recover the cells by shaking at 37 °C.    Another rich medium can substitute for the recovery.
    The recovery time varies with the antibiotic selection.
    Tetracycline: 60-120 minutes
     Chloramphenicol: 60-120 minutes
    8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate.
    Use glass beads to spread the cells.
    9) Count colonies after incubation.
    • Transform:
    • Constitutive Promoter
      • BBa_J23108: Constitutive Promoter from Anderson's Library
        • Resuspended DNA from 2014:4:4C
        • pSB1C3 Chloramphenicol resistance
    • BBa_J45199: Banana generator without promoter
      • Resuspended DNA from 2013:4:6I
      • pSB1C3 Chloramphenicol resistance
      • Requires promoter and 5mM isoamyl alcohol
    Protocol: Heat Shock Transformation of E. coli
    1) Place 20 ul of cells in a pre-chilled Eppendorf tube.
    2) Add 2 ul of DNA to the chilled cells
    3) Mix gently by flicking the tube.
    4) Chill on ice for 10 minutes.
    5) Heat shock at 42 °C for 30 seconds.
    6) Return to ice for 2 minutes.
    7) Add 200 ul LB medium and recover the cells by shaking at 37 °C.    Another rich medium can substitute for the recovery.
    The recovery time varies with the antibiotic selection.
    Tetracycline: 60-120 minutes
     Chloramphenicol: 60-120 minutes
    8) Plate out 20uL of the cells on selective LB. And 200 uL on another plate.
    Use glass beads to spread the cells.
    9) Count colonies after incubation.
    • Plate fresh transformations:
      • BBa_R0040 LBA+Cm
      • BBa_J45119 LBA+Tet
      • BBa_J45199 LBA+Cm
    • Streak NEB Turbo in LBA for singles (tomorrow set an overnight for competent cell production).
    • Send email asking for salicylic acid and isoamyl alcohol.

    8/07 Single colonies from Transformations

    Done

    8/07 Minipreps

    Done

    8/07 Digestion from miniprep set up

    Digestion:


    FD Restriction Digestion
    Reagent Volume (uL)
    NF ddH2O
    17
    Buffer
    3
    FD Enzyme I
    1
    FD Enzyme II
    1
    DNA
    3
    Total Volume
    25


    Cut pPB.014 with SpeI & PstI
    Expected fragments:
    1. ~2 kb
    2. ~18 bp
    Cut pPB.004 with XbaI and PstI
    Expected fragments:
    1. ~ 2 kb
    2. 1.765 kb

    8/07 SAGE set up


    Sage:
    SAGE on thick gel (3 mm) 1% agarose should be fine.
    There should be two bands for pPB.004.

    Gel extraction:
    1. Cut out the second band of pPB.004 and pPB.005 (1.8 kb and 1.3 kb respectively).
    2. Weigh the agarose fragments (100 mg gel : 300 uL QG Buffer)
      • Max amount is 400 mg
    3. Solubilize agarose completely by incubating at 50ºC for 10 min in water bath. Vortexing the tube every 3 minutes helps.
    4. Color of mixture should be yellow. Otherwise add 10 uL of 3 M sodium acetate pH 5.
    5. Add the equivalent of 1 gel volume of isopronanol and mix the tube (although for fragments between 500 bp and 4 kb this should matter).
    6. Place column in a 2 mL collection tube
    7. Load sample into column. Centrifuge 1 min @ 13 000 rpm.
    8. Discard flowthrough and repeat adding 0.5 mL of QG buffer.
    9. Discard flowthorugh and wash with 0.75 mL of PE Buffer. Let the column sit for 5 min prior centrifugation.
    10. Discard flowthrough and centrifuge again to ensure all residual ethanol is gone.
    11. Place the column into a clean 1.5 mL Epp and elute by adding 50 uL of NF-ddH2O. Make sure to wait 2 min before centrifugation.
    12. Store at 4ºC overnight or -20ºC for longer periods.

    Ligation set up

    Ligation:
    1. Fermentas T4 DNA Ligation
      Reagent
      Vector (pPB.003)
      		 25 ng
      Insert (pPB.014)
      		 75 ng
      T4 DNA Ligase Buffer 10X
      		 1 uL
      T4 DNA Ligase
      		0.5 uL
      NF-ddH2O
      		 as needed
      Total
      		 10 uL

    2. Incubate vector @22ºC for 60-120 minutes and insert for 0.5-1 hour.
    3. Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
    4. Recover for 60 minutes on LB.
    5. Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
    • Recovered the cells for 45 minutes: No colonies (27/06)
    • Re-plated remaining recovered cells (overnight @ room temperature): No colonies (27/06)
    • Repeated transformation (29/06): No colonies.

    pPB.016 Enhanced Wintergreen Generator

    • pPB.015: Enhanced Wintergreen generator Salis Promoter (BBa_23108)
      • Requires 2 mM Salicylic acid

    8/07 Digestion from miniprep set up

    FD Restriction Digestion
    Reagent Volume (uL)
    NF ddH2O
    17
    Buffer
    3
    FD Enzyme I
    1
    FD Enzyme II
    1
    DNA
    3
    Total Volume
    25


    Cut pPB.014 with SpeI & PstI
    Expected fragments:
    1. ~2 kb
    2. ~18 bp
    Cut pPB.005 with XbaI and PstI
    Expected fragments:
    1. 3.4 kb
    2. 1.26 kb


    8/07 SAGE set up


    Sage:
    SAGE on thick gel (3 mm) 1% agarose should be fine.
    There should be two bands for pPB.005.

    Gel extraction:
    1. Cut out the second band of pPB.004 and pPB.005 (1.8 kb and 1.3 kb respectively).
    2. Weigh the agarose fragments (100 mg gel : 300 uL QG Buffer)
      • Max amount is 400 mg
    3. Solubilize agarose completely by incubating at 50ºC for 10 min in water bath. Vortexing the tube every 3 minutes helps.
    4. Color of mixture should be yellow. Otherwise add 10 uL of 3 M sodium acetate pH 5.
    5. Add the equivalent of 1 gel volume of isopronanol and mix the tube (although for fragments between 500 bp and 4 kb this should matter).
    6. Place column in a 2 mL collection tube
    7. Load sample into column. Centrifuge 1 min @ 13 000 rpm.
    8. Discard flowthrough and repeat adding 0.5 mL of QG buffer.
    9. Discard flowthorugh and wash with 0.75 mL of PE Buffer. Let the column sit for 5 min prior centrifugation.
    10. Discard flowthrough and centrifuge again to ensure all residual ethanol is gone.
    11. Place the column into a clean 1.5 mL Epp and elute by adding 50 uL of NF-ddH2O. Make sure to wait 2 min before centrifugation.
    12. Store at 4ºC overnight or -20ºC for longer periods.

    Ligation set up

    Ligation:
    1. Fermentas T4 DNA Ligation
      Reagent
      Vector (pPB.014)
      		 25 ng
      Insert (pPB.005)
      		 75 ng
      T4 DNA Ligase Buffer 10X
      		 1 uL
      T4 DNA Ligase
      		0.5 uL
      NF-ddH2O
      		 as needed
      Total
      		 10 uL

    2. Incubate @22ºC for 10 minutes.
    3. Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
    4. Recover for 60 minutes on LB.
    5. Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
    • No colonies.



    Check-up after vacations:


    2/09/14:
    • Run gel of old samples
      • pPB.003 TetR Promoter
      • pPB.004 RBS-ATF1
      • pPB.005 RBS-BMST1
      • pPB.014 BBa_J23108
    SAGE showed very faint bands suggesting a low [DNA] ( I used 4 uL of QIAGEN gel purification products).
    • Start 5 mL overnights of single colonies from Estefania’s plates.
      • pPB.015: Anderson’s Promoter + BMST1
      • pPB.016: Anderson’s Promoter + ATF1
      • pPB.022 (BBa_J45004) : BMST1 from Registry 2012 kit
    3/09/14:

    The gel purified fragments I have should be:
    1. pPB.003 (Digested with SpeI & PstI) ~2.1 kb
    2. pPB.004 (Digested with XbaI & PstI) ~1.765 kb
    3. pPB.005 (Digested with XbaI & PstI) ~1.26 kb
    4. pPB.014 (Digested with SpeI & PstI) ~2 kb
    5/09/14:

    Analytical digestion of Estefania’s pPB.015 and pPB.016 with NheI and PstI.

    The Anderson’s promoter has two NheI sites. A restriction digestion with NheI and PstI should yield the following fragments:

    pPB.015: 27 bp, 1.2 kb, 2.06 kb
    pPB.016: 27 bp, 1.11 kb, 2.06 kb

    FD Restriction Digestion
    Reagent Volume (uL)
    NF ddH2O
    17
    Buffer
    3
    FD NheI
    1
    FD PstI
    1
    DNA
    3
    Total Volume
    25

    Incubate at 37ºC for at least 1 hour.

    Ligation setup:
    1. Fermentas T4 DNA Ligation
      Reagent
      Vector (pPB.003 or pPB.014)
      		 25 ng
      Insert (pPB.004 or pPB.005)
      		 75 ng
      T4 DNA Ligase Buffer 10X
      		 1 uL
      T4 DNA Ligase
      		0.5 uL
      NF-ddH2O
      		 as needed
      Total
      		 10 uL

    2. Incubate @22ºC for 10 minutes.
    3. Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
    4. Recover for 60 minutes on LB.
    5. Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
    • No colonies.
    Banana Fix

    The sequencing results from 16/09/2014 show that the promoter got inserted between the EcoRI and XbaI restriction sites. It is supposed to be downstream of XbaI. Also, the promoter sequence seems to be erroneous on the assembled plasmid (pPB.016) but not on the plasmid containing the promoter (pPB.014). I will clone it again.
    1.- Restriction digestion

    Cut 300 ng of pPB.014 with SpeI & PstI
    Expected fragments:
    1. ~2 kb
    2. ~18 bp
    Cut 300 ng of pPB.004 with XbaI and PstI
    Expected fragments:
    1. ~ 2 kb
    2. 1.765 kb
    FD Restriction Digestion
    Reagent Volume (uL)
    NF ddH2O
    13.5
    Buffer
    4
    FD Enzyme I
    1.5
    FD Enzyme II
    1.5
    DNA
    3
    Fast AP 1.5 uL


    Incubate at 37ºC for at least one hour.

    pPB.004 Digestion Product has to be gel purified. Extract the lower band of the gel.
    pPB.014 Purify with PCR cleanup kit.

    2.- Purification

    Run a gel with a lot of pPB.004 (~20 uL of digestion product) for purification using gel excision kit. Purify pPB.014 with the PCR cleanup kit.

    3.- Ligation
    1. Fermentas T4 DNA Ligation
      Reagent
      Vector (pPB.003)
      		 25 ng
      Insert
      		 75 ng
      T4 DNA Ligase Buffer 10X
      		 1 uL
      T4 DNA Ligase
      		0.5 uL
      NF-ddH2O
      		 as needed
      Total
      		 10 uL

    2. Incubate @22ºC for 10 minutes.
    3. Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
    4. Recover for 60 minutes on LB.
    5. Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.
    4.- Sequencing


    September 17,2014
    The sequenced samples from Estefania (EST pPB.015) do not contain matching repeats to the reference sequence (pPB.015). However, the old samples I was working with (pPB.005) have a match with the original mint construct taken from the registry kit. In other words, the pPB.005 contains a BMST1 CDS without promoter, and pPB.015 has a non matching sequence (corresponds to limonene enzyme…). My guess is that the wrong samples were sent for sequencing due to mislabeling or magic.



    September 23, 2014
    Fix it.
    I digested some pPB.014 (Fig.1) with SpeI and PstI. This will open up a cohesive end downstream of the BBa_J23108 promoter. Alkaline phosphatase is added to the reaction mix to dephosphorylate the 5’ end of the fragment, this will help to prevent vector re-circularisation. The ATF1 promoter-less generator will be cut out from pPB.004 by restriction digestion with XbaI and PstI. Complementary cohesive ends will be formed upstream of the BBa_B0030 RBS (XbaI) and downstream of the BBa_0012 transcriptional terminator (PstI). This fragment (Fig.2: 1) will be inserted into the linearised pPB.014 (Fig.2: 4) by ligation with T4 DNA ligase after a gel purification.The resulting plasmid will contain the promoter upstream of the RBS, enabling constitutive expression of ATF1, and thus production of banana smell when transformed cells are grown in presence of isoamyl alcohol.

    Fig.1: Constitutive promoter from the Anderson’s promoter library (BBa_J23108) in a pSB1C3 vector with standard BioBrick format.


    Fig.2: Restriction digestion products:
    1.- RBS (BBa_B0030), ATF1 CDS, and transcriptional terminators (BBa_B0010 and BBa_B0012). This will be purified by gel excision.
    2.- Fragment of pSB1C3 vector.
    3.- Fragment between SpeI and PstI sites (BioBrick suffix)
    4.- Linearised pSB1C3 with appropriate cohesive ends for inserting fragment 1.

    Fig.3: Final assembled plasmid containing a constitutive ATF1 expression system in a pSB1C3 vector with standard BioBrick format.




    gBlock PCR Troubleshooting


    Reagent Volume
    1x 4x
    Nuclease-free water 71 ul 284 ul
    5x Phusion HF Buffer 20 ul 80 ul
    10 mM dNTPs 2 ul 8 ul
    Forward Primer (10 uM) 1 ul 4 ul
    Reverse Primer (10 uM) 1 ul 4 ul
    Template DNA (Plasmid, 1:10 dilution) 1 ul 4 ul
    DMSO 3 ul 12 ul
    Phusion DNA Polymerase 1 ul 4 ul
    Total Volume 100 ul

    Thermocycler Protocol: NEB Phusion
    Temp Time
    Start 98 °C 30 sec Melt
    Cycle 1 98 °C 10 sec Melt
    35 Cycles
    Cycle 2 52 °C 30 sec Anneal
    Cycle 3 72 °C 40 sec Extend
    Finish 72 °C 5 min Extend
    Store 10 °C Forever Store



    LIGATE EVERYTHING
    Ligation

    [pPB.004]= 8.5 ng/uL
    [pPB.014]= 2.6 ng/uL

    Banana and Mint ligations:

    pPB.004 (XbaI PstI) + pPB.014 (SpeI PstI)
    pPB.005 (XbaI PstI) + pPB.014 (SpeI PstI)

    Controls:
    Intact pPB.014
    pPB.014 (SpeI PstI)
    Ligase + pPB.014 (SpeI PstI)
    Ligase + NF dH2O


    Digested 30 ng of gPB.005 and 30 ng of gPB.006 with EcoRI and PstI. Both of them were column purified using the QIAGEN kit. They were eluted in 10 uL of NF dH2O. Some linearised pSB1C3 was prepared by PCR with oPB.081 and oPB.082, the product was column purified. A restriction digestion of pPB.003 with EcoRI and PstI was done in parallel and gel purified.

    [gPB.005]= 2.2 ng/uL
    [gPB.006]= 2.3 ng/uL


    gBlocks Mint and Geosmin:

    gPB.003 (EcoRI PstI) + pSB1C3 (EcoRI PstI)
    gPB.005 (EcoRI PstI) + pSB1C3 (EcoRI PstI)
    gPB.006 (EcoRI PstI) + pSB1C3 (EcoRI PstI)
    PCR Vector:
    Controls:
    Intact pSB1C3 (PCR Product)
    pSB1C3 (EcoRI PstI)
    Ligase + pSB1C3 (EcoRI PstI)
    Ligase + NF dH2O


    1. Keep insert+vector volume below 4 uL.
      Fermentas T4 DNA Ligation
      Reagent
      Vector (pPB.003)
      		 25 ng
      Insert
      		 75 ng
      T4 DNA Ligase Buffer 10X
      		 1 uL
      T4 DNA Ligase
      		0.5 uL
      NF-ddH2O
      		 as needed
      Total
      		 10 uL

    2. Incubate @22ºC for 10 minutes.
    3. Transform 20 uL of NEB Turbo competent cells with 5 uL of ligation product.
    4. Recover for 60 minutes on LB.
    5. Plate on selective LBA+Cm plates. Incubate at 37ºC, colonies should appear overnight or within 24 hours max.

    The first 6 samples are PCR products of gPB.005 (BMST1) with JW.340 and JW.341. One uL of 1:10 dilution from the gBlock stock was used as template for the first set of three samples (~1 ng of DNA). The volume of 10 uM primers was 1, 1, and 0.5 uL. One uL directly from the stock was used as template for the second set (~10 ng of DNA). The volume of 10 uM primers was 0.5, 1, and 1.5 uL respectively. The last two lanes before the ladder are the PCR products of 1uL of BBa_J45119 (~140 ng) with oPB.081 and oPB.082. I got confused and thought this biobrick was in pSB1C3 vector, but I was wrong. I expected a band of ~ 2 kb, instead there is bands of ~3.3 kb which correspond to the length of pSB1AT3.

    The second half of the gel follow the same scheme as the first one except that the DNA template is gPB.006 (Geosmin) and the vectors are from a 1:10 dilution of a 140 ng/uL BBa_J45119 mini prep (~14 ng) instead. The used program is:
    Thermocycler Protocol: NEB Phusion
    Temp Time
    Start 98 °C 30 sec Melt
    Cycle 1 98 °C 10 sec Melt
    35 Cycles
    Cycle 2 52 °C 30 sec Anneal
    Cycle 3 72 °C 60 sec Extend
    Finish 72 °C 10 min Extend
    Store 10 °C Forever Store





    -


    I will repeat the PCR for the gPB.003 (    oPB.013 and oPB.014), gPB.005, gPB.006 (JW.340 and JW.341) using the following conditions:

    Phusion MasterMix
    Reagent Volume (uL)
    DNA 1
    Fwd (10 uM) 2.5
    Rev (10 uM) 2.5
    DMSO 1.5
    MM 25
    H2O 17.5
    50

    I will do each sample by duplicate varying only the [DNA template] using 1 uL directly from stock tubes and a 1:10 dilution as well. For the pSB1C3 (oPB.081 and oPB.082) I will use 1 uL of pPB.003 (RBS) or pPB.004 (Banana generator) at 1:10 and 1:100 dilutions.

    The program will be the same except that I will increase the annealing temperature to 55ºC.

    gPB.003: 1307 bp
    gPB.005: 1211 bp
    gPB.006: 1238 bp
    pSB1C3: 2070 bp

    The PCR was repeated with a 58ºC annealing temperature:







    29/09/2014

    Colony PCR of pPB.015 and pPB.016 BL21 colonies:

    Phusion MasterMix Final concentration
    Reagent Volume (uL)
    DNA
    1
    < 250
    Fwd (10 uM)
    1
    0.5 uM
    Rev (10 uM)
    1
    0.5 uM
    DMSO
    0.6
    3%
    MM
    10
    1X
    H2O
    6.4
    20


    Purification/ligation of digested gBlocks:

    Keep insert+vector volume below 4 uL.
    Fermentas T4 DNA Ligation
    Reagent
    Vector (pPB.003)
    		 25 ng
    Insert
    		 75 ng
    T4 DNA Ligase Buffer 10X
    		 1 uL
    T4 DNA Ligase
    		0.5 uL
    NF-ddH2O
    		 as needed
    Total
    		 10 uL



    Digestion of gBlocks:

    Digestion of vectors
    Plamid DNA
    20
    10x FD Buffer
    10
    FD Enzyme I
    2.5
    FD Enzyme II
    2.5
    FastAP
    2.5
    NF H2O
    62.5
    Total
    100
    Incubate @ 37ºC for 2-4 hours with shaking.
    Digestion of inserts
    Plamid DNA
    16
    10x FD Buffer
    2
    FD Enzyme I
    1
    FD Enzyme II
    1
    NF H2O
    0
    Total
    20
    Incubate @ 37ºC for 0.5-1 hours with shaking.

    Hurrying
    Heat shock:
    5 uL of ligation product to 20 uL of NEB Turbo competent cells @42°C for 30 seconds.
    2 min in ice.
    200 uL of LB and recover for a couple of hours @ 37ºC.

    *Prepare NEB Turbo overnight culture for electroporation in cae heat shock fails.

    Colony PCR 2:

    Phusion MasterMix Final concentration
    Reagent Volume (uL)
    DNA
    1
    < 250
    Fwd (10 uM)
    1
    0.5 uM
    Rev (10 uM)
    1
    0.5 uM
    DMSO
    0.6
    3%
    MM
    10
    1X
    H2O
    6.4
    20

    Thermocycler Protocol: NEB Phusion
    Temp Time
    Start 98 °C 30 sec Melt
    Cycle 1 98 °C 10 sec Melt
    35 Cycles
    Cycle 2 55 °C 30 sec Anneal
    Cycle 3 72 °C 60 sec Extend
    Finish 72 °C 10 min Extend
    Store 10 °C Forever Store

    *Colony PCR pics are unavailable due to failure of USB key (files were corrupted and pictures became black).

    1/10/2014
    Submerged agarose gel electrophoresis of the PCR products showed a ~1.3 kb band on the sample from colony#2 (14+4). The expected product with oPB.006 and oPB.074 is ~2 kb. Phusion polymerase extends at a rate of ~ 100 bp/s, so the 60 s extension time should be good. However, the primers have a Tm of 51.8ºC and 49.7ºC respectively. Therefore, the annealing temperature might be too high. Run another colony PCR with the verification primers using an annealing temperature of 50.7ºC.

    Thermocycler Protocol: NEB Phusion
    Temp Time
    Start 98 °C 30 sec Melt
    Cycle 1 98 °C 10 sec Melt
    35 Cycles
    Cycle 2 50 °C 30 sec Anneal
    Cycle 3 72 °C 60 sec Extend
    Finish 72 °C 10 min Extend
    Store 10 °C Forever Store


    October
    1/10/2014

    pPB.015 (Mint) and pPB.016 (Banana) minipreps:

    I used the Promega kit.


    pSB1C3 and gBlock amplification by PCR using Phusion polymerase:

    Thermocycler Protocol: NEB Phusion
    Temp Time
    Start 98 °C 30 sec Melt
    Cycle 1 98 °C 10 sec Melt
    35 Cycles
    Cycle 2 59 °C 30 sec Anneal
    Cycle 3 72 °C 60 sec Extend
    Finish 72 °C 10 min Extend
    Store 10 °C Forever Store


    2/10/2014

    To DO:
    1.- Colony PCR with oPB.006 & oPB.074, annealing temperature= 50ºC.
    2.- Nanodrop miniprep samples.
    3.- Set analytical digestion of pPB.015 and pPB.016 with AvrII, BstXI, HindIII, and PstI.
    Expected fragments:
    • 300 bp
    • 968 bp
    • 1258 bp
    • 2076 bp
    4.- Send for sequencing if positive.
    5.- gBlock PCR again.
    6.- Set up electroporation stuff.
    7.- Gel purify yesterday’s PCR products.
    8.- Run a small volume SAGE of purified PCR products to confirm size.
    9.- Digest old PCR products.
    10.- Purify digestion products.
    11.- Set up ligation reaction.
    12.- Electroporate/Heat shock.

    Protocol: E. coli Chromosomal Integration (à la Datsenko and Wanner)
    Preparation of Electrocompetent Cells
    Note: Competent cells should never be vortexted, as this will cause them to lyse and release salts into the media. Resuspend cells by pipeting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.
    1) The night before the transformation, start an overnight culture of cells.
    5 ml LB Amp.
    2) The day of the transformation, dilute the cells 100X.
    100 ml LB Amp.
    Grow at 30°C for about 90 minutes.
    3) Harvest the cells.
    When the cells reach an OD600 of between 0.6 and 0.8.
    Split the culture into 2x 50 ml falcon tubes, on ice.
    Centrifuge at 4 °C for 10 min at 4000 rpm.
    4) Wash and combine the cells.
    Remove the supernatant.
    Resuspend the cells in 2x 25 ml of ice cold water.
    Combine the volumes in a single 50 ml falcon tube.
    5) Wash the cells 2 more times.
    Centrifuge at 4 °C for 10 min at 4000 rpm.
    Resuspend in 50 ml of ice cold water.
    Repeat.
    6) Wash and concentrate the cells for electroporation.
    Centrifuge at 4 °C for 10 min at 4000 rpm.
    Resuspend in 1-2 ml of ice cold water.
    We will use 200 ul of washed cells per transformation.

    Dialysis of PCR or Digestion Products
    Note: DNA for electroporation must be free of salts to avoid arcing.
    1) Float a filter in a Petri dish filled with water.
    Millipore membrane filter 0.025 uM.
    2) Pipet one drop of PCR product onto the filter.
    200 ng is needed per transformation.
    20 - 100 ul fits well on one filter.
    3) Collect the drop after 30 - 45 minutes.
    The volume will change, but the DNA is not lost.
    Protocol: Electroporation of E. coli
    Note: This protocol uses 200 ul of cells and 200 ng of DNA.
    The cuvette is 2 mm. We use electroporation setting EC2.
    A time constant above 5 indicates a successful electroporation.
    1) Prepare BD tubes with a pipette filled with LB at the interior of each tube (pipette supplied with the electroporation cuvette)
    2) Test the purity of the electrocompetent cells.
    Add 200 ul of washed cells to a cuvette.
    3) Mix the cells and DNA in a cuvette.
    200 ul of washed cells with 200 ng of PCR product.
    Keep the cuvette on ice until just before the electroporation.
    4) Preload a pipette with 1 ml of LB.
    5) Pulse the cuvette with voltage.
    Dry the electrodes with a kimwipe prior to loading.
    Use the EC2 setting.
    6) Listen for arcing.
    A cracking sound means all the cells are dead.
    Note the time constant: 5 is good, 5.8 is great.
    7) Immediately recover the cells.
    Add the 1 ml of preloaded LB and pipet up and down to mix.
    Collect 1 ml of cells, some volume is lost in the cuvette.
    8) Incubate 2 h at 37 °C with shaking.
    9) Plate 100 ul of recovered cells on selective plates.
    Use antibiotic appropriate to the part being integrated.
    Let the other 900 ul rest overnight at room temperature.
    10) Concentrate and plate the remaining cells
    Spin down quickly and resuspend in 100 ul LB before plating.
    Transformed cells should be incubated at 37 °C.
    Colonies should appear 24-48 h after plating.
    13.- Recover.
    14.- Plate.


    Start again
    October 8, 2014

    Banana BioBrick
    [DNA] (ng/uL) Volume (uL) Amount (ng)
    vector pPB.014
    34
    1
    34
    insert (pPB.004)
    15
    5
    75
    Buffer
    2
    T4 Ligase
    1
    9
    H2O
    11
    Jasmine gBlock
    [DNA] (ng/uL) Volume (uL) Amount (ng)
    vector
    4
    12
    48
    insert (pPB.003)
    5
    19
    92.4
    1307
    Buffer
    6
    T4 Ligase
    3
    40


    gPB.003 PCR with oPB.013 & oPB.014. pSB1C3 PCR with oPB.081 and oPB.082.

    Phusion MasterMix Final concentration
    Reagent Volume (uL)
    DNA
    1
    < 250
    Fwd (10 uM)
    1
    0.5 uM
    Rev (10 uM)
    1
    0.5 uM
    DMSO
    0.6
    3%
    MM
    10
    1X
    H2O
    6.4
    20

    Thermocycler Protocol: NEB Phusion
    Temp Time
    Start 98 °C 30 sec Melt
    Cycle 1 98 °C 10 sec Melt
    35 Cycles
    Cycle 2 59 °C 30 sec Anneal
    Cycle 3 72 °C 60 sec Extend
    Finish 72 °C 10 min Extend
    Store 10 °C Forever Store


    Yesterday’s heat shock transformation of E. coli tnaA deletion mutant from Keio Collection with geosmin, JMT, BMST1, and ATF1 expression cassettes yielded colonies for all except ATF1.

    Colony PCR

    Phusion MasterMix Final concentration
    Reagent Volume (uL)
    DNA
    1
    < 250
    Fwd (10 uM)
    1
    0.5 uM
    Rev (10 uM)
    1
    0.5 uM
    DMSO
    0.6
    3%
    MM
    10
    1X
    H2O
    6.4
    20

    Expected product with oPB.006 and oPB.074 is ~2 kb. Phusion polymerase extends at a rate of ~ 150 bp/s, so the 60 s extension time should be good. The primers have a Tm of 51.8ºC and 49.7ºC respectively.

    Thermocycler Protocol: NEB Phusion
    Temp Time
    Start 98 °C 30 sec Melt
    Cycle 1 98 °C 10 sec Melt
    35 Cycles
    Cycle 2 50.7 °C 30 sec Anneal
    Cycle 3 72 °C 60 sec Extend
    Finish 72 °C 10 min Extend
    Store 10 °C Forever Store


    *Geosmin pPB.025 was successfully transformed.


    Characterizations planning
    Media Preparation:
    M9 Minimal Media 0.4% Glucose. Bring to 1 L
    Reagent Qty
    5x M9 Salts 200 mL
    1M MgSO4 2 mL
    20% Glucose Stock Solution
    		 20 mL
    1M CaCl2 0.1 mL


    Complete AA mix 2 g

    Banana:
    5 mM isoamyl alcohol
    Molecular Weight 88.15
    Add 4.4075 g to 100 mL of sterile dH2O. This is a 1 M stock solution.
    Use 50 uL in 9.95 mL of M9 Media.

    Mint:
    2 mM Salicylic acid
    Molecular Weight 138.12
    Add 6.906 g to 100 mL of dH2O. This is a 1 M stock solution.
    Use 20 uL in 9.98 mL of M9 Media.

    Geosmin:
    FPP
    Jasmine:
    Jasmonic acid
    Limonene:
    FPP
    Butter:
    Acetoin

    1.- Make an overnight culture.
    2.- Dilute them 1:200 by duplicate.
    3.- Treat one of the duplicates with substrate, leave the other without, and grow to exponential phase. (Make a control of selective media+substrate)
    4.- Smell them when they reach mid-log phase.
    5.- Add substrate to the untreated samples.
    6.- Grow to stationary phase and smell.

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