Notebook

2022/08/16

Kazuma Tsurumaki

  1. Agarose gel electrophoresis

    Produced agarose gel & ran gel electrophoresis to verify the pgem-vector. used a nicked pgem-vector.

2022/09/08

Kazuma Tsurumaki

  1. Dissolved parts

    Dissolved glu-lac1,glu-lac2, and cell-glu2 in Milli-Q. 10ng/μl of parts are made. cell-glu1 is on order.

  2. Digest a plasmid with HindIII Used HindIII to cut a plasmid.

    HindIII:0.4㎕ pTWV228(0.5㎍/㎕,4039bp) :1㎕ NEB Buffer: 1㎕ MiliQ:7.6㎕

    pTWV228 cut with HindIII will be 0.019pmol/μg

    The cleavage product is named “pTWV228” and frozen.

2022/09/11

Kazuma Tsurumaki, Kota Toyoda, Gonzalo Sifuentes

  1. Gibson assembly According to the protocol offered by NEW England Biolabs

    glc-ace1- 3㎕ glc-ace2- 3㎕ vector-  0.3㎕ master mix(2x) 10㎕ milliQ 3.7㎕

    named the product “gibson 9/11”

  2. Made LB broth

    made LB agar medium with ampicillin and liquid medium. Both were autoclaved.

2022/09/14

Kazuma Tsurumaki

  1. Transformation gibson 9/11 :1μl Competent cells:50μl

    Also, transformed 1μl of vector to use as a positive control. Following the Single Tube Transformation Protocol provided by iGEM, except for the process of heat shock.

    heat-shock for 30sec at 42°C on a heat block, according to Mr. Tashiro, working in the synbio lab at Kyushu Univ, who offers us cells.

  2. Made competent cells According to this site
    https://www.gene.mie-u.ac.jp/Protocol/Original/Transform-Comp-Cell-Freeze.html

    Protocols

    Shake at 37℃ to culture. Dispense into tubes . Incubate on ice for 30min. Centrifuge at 2500rpm at 4℃ for 5min . Discard mediums. Add 25 ml of 50 mM CaCl2 and suspend on ice. Incubate on ice for 60min. Centrifuge at 2500rpm at 4℃ for 5min. Discard supernatant. Add 5ml of 50mM CaCl2 / 20% Glycerol and suspend. Dispense 220 µl into one tube and quench at -80℃.

    BL21(DE3) is being cultured in 5ml LB medium.

2022/09/15

Kazuma Tsurumaki, Kota Toyoda

  1. Transformation checked yesterday’s plates

    Positive control: many colonies, Insert : 0 colonies,

    Therefore, the transformation process was successful, but the INSERT plasmid was not present. We” ll try another concentration of DNA in gibson assembly.

  2. Inactivation of restriction enzyme heat-shocked cleavage products cut with HindIII at 70℃ for 15min.

  3. Gibson assembly again

    glc-ace1: 4㎕, glc-ace2: 4㎕, vector: 0.5㎕, master mix(2x): 10㎕ milliQ: 1.5㎕

    named the product “gibson 9/15”

  4. Made competent cells

    OD600: 0.9

    This is lower than expected.

    Cultured in centrifuge tubes

    LB 50ml, Bacterial solution(cultured from 20220914) 1ml

2022/09/16

Kazuma Tsurumaki, Kota Toyoda

  1. Determined whether “gibson 9/15” was assembled as designed
  1. Nanodrop

    gibson 9/15 DNA Concentration: 1360ng/㎕ 260/280:2.20

    Overall, we determined that gibson 9/15 is in a plasmid state.

  2. Made competent cells

    made successfully. frozen at -80℃.

2022/09/20

Kazuma Tsurumaki

  1. Gibson assembly

    aatA2:3μl, poxB:3μl, vector:1.5μl, miliQ:2.5μl, mastermix:10μl,

    named the product “gib9/20”

  2. Transformation

    Transformed gib9/20 and con9/15 into competent cells we made. Made 2 versions. “Low-concentration version” is transformed with DNAs as they are. “High-concentration version” is transformed with DNAs centrifuged and the supernatant removed. We” ll check at 9:00 AM, tomorrow.

20220921

Kazuma Tsurumaki, Kota Toyoda

  1. Transformation observed colonies (9:40)

    Number of colonies: insert(High concentration):6 insert(Low concentration):1 control(High concentration):many  control(Low concentration):85 (¼ of area)

    Picked colonies inoculated it into LB-Cam and cultured overnight.

2022/09/22

Kazuma Tsurumaki

  1. Measured OD600 of cam cultured from yesterday

    OD600: 0.416 at 10:10

  2. Dispended IPTG 5 g of IPTG dissolved in 21 ml of water. IPTG(1mol/l) was dispensed in 1.4ml portions and stored frozen.

  3. IPTG induction and culturing Added 1.5μL IPTG and ampicillin to LB medium and cultured with 30㎕ of the culture fluid of gib9/20. at the same time, cultivated without IPTG as control

Procedure

Add 1mol/L IPTG and ampicillin(100x) to 1.5ml of LB medium Add 30μl of the culture fluid to the LB medium. Cultured for 4-5h at 37℃ in culture tubes in a shaking water bath.

Result: OD600

Since the values at the 1-hour-point was too different, we” ll carried out the same experiment.

  1. Prepared standard curve for acetic acid determination According to the protocol, made 20,16,12,8,4,0mM acetate, and measured absorbance. Protocol: https://www.bioassaysys.com/datasheet/EOAC.pdf
    To measure absorbance, used an absorbance spectrophotometer
    (USHIO Picoscope https://www.ushio.co.jp/jp/technology/lightedge/201708/500214.html)
    Measured absorbance of 455-630nm. However, because the data is too irregular, we” ll try again tomorrow. This might be because we failed to centrifuge samples to remove particles.

2022/09/23

Kazuma Tsurumaki, Kota Toyoda

  1. Prepared standard curve for acetic acid determination

    According to the protocol, made 0,4, 8, 12, 16, and 20 mM acetate standards in water, and made a standard curve. To remove particles, centrifuge the samples and use the supernatant as a test object.

  2. Miniprep

    Following Promega's protocol, miniprep 2 ml of the concentrated medium cultured yesterday. Named the product “9/23 Plasmid”.

2022/09/24

Kazuma Tsurumaki

  1. IPTG induction and cultured

    Added 1.5μl of IPTG(1mol/l) and ampicillin(100x) to 1.5ml of LB medium and cultured with 30㎕ of cultured fluid from 13:30 just like 20220922. Measured od600 and concentration of acetate.

    Result:

  2. Analyzed 9/23 Plasmid

    Nanodrop: 33ng/㎕ 260/280:1.77

  3. Digested 9/23 Plasmid with EcoRI

    EcoR1 1㎕ CutSmartbuffer 5㎕ 9/23 Plasmid 20㎕ Milli-Q 24㎕

2022/09/26

Kazuma Tsurumaki, Kota Toyoda, Gonzalo Sifuentes, Akito Shima

  1. IPTG induction and cultured

    Prepared 3ml of LB medium containing 0mM,0.5mM,1mM,1.5mM of IPTG and cultured E.Coli picked at 20220921 in it, followed to 20220922 - IPTG induction and culturing - procedure for 5 hours. Measured concentration of acetate and OD600.

Result:

Since the amount of reagent was wrong in the third hour and there was a significant discrepancy, the test will be conducted again tomorrow.

20220928

Kazuma Tsurumaki, Kota Toyoda

  1. Made standard carve of to assay concentration of acetate

    Prepared 0,4, 8, 12, 16, and 20 mM acetate standards in LB Measured concentration according to the protocol by the acetate assay kit

  2. Gibson assembly

    master mix(2x):10㎕ cell-glu1:3㎕ cell-glu2:3㎕ vector:2㎕ H2O:2㎕ Named the product 9/28 cell gib

  3. IPTG induction and cultured again

    OD600 and the concentration of acetate were too low.

2022/09/29

Kazuma Tsurumaki

  1. Transformation

    9/28 cell gib: 1㎕ Competent cell: 50 ㎕ Transformed with DNAs centrifuged and the supernatant removed. We'll centrifuge DNAs from now on before transformation and will not make a “Low-concentrate version”

  2. IPTG induction and cultured again same as 20220922

    Reduced IPTG: 0,1mM. We” ll check the pH of the medium tommorow.

2022/09/30

Kazuma Tsurumaki

  1. Transformation Checked mediums made yesterday No colony is observed. The same experiment will be conducted.

  2. Measured pH of the medium Cultured overnight pH: 8

  3. IPTG induction and cultured again the same as 20220922 Only the concentration of IPTG was differ from the procedure of 20220922

    IPTG: 0,1 mM

    pH test will be conducted tomorrow.

2022/10/01

Kazuma Tsurumaki, Kota Toyoda

  1. pH of the medium with acetogenic bacteria

    pH: 8-9 (both 0mM and 1mM IPTG)

  2. Gibson assembly According to the protocols offered by NEB.

    glc-ace1:2:8 ㎕, vector:3 ㎕, milliQ:1 ㎕, mastermix:20 ㎕,

    Named the product “10/1 cell”

  3. Transformation Transformed “9/28 cell gib” and control on each plate since 16:05. We” ll check whether the colony appears or not tomorrow.

2022/10/02

Kazuma Tsurumaki, Kota Toyoda, Gonzalo Sifuentes

  1. Checked plates No colony was observed

  2. Restriction Enzyme Treatment of Vectors Once More named the product “V”

  3. Gibson assembly

    glc-ace1:3.5㎕, glc-ace2:3.5㎕, vector 2.5㎕, milliQ:0.5㎕, master mix(2x):10㎕,

    Named the product “gib10/02”

  4. Transformation Transformed gib10/02 and control on each plate Both insert and control are cultured in each Petri plate. The Petri plates will be checked tomorrow.

2022/10/03

Kazuma Tsurumaki

  1. Checked the Petri plates. No colony is observed.

  2. Transformation Transformed gib10/02 and control.

Competent cells: 50 ㎕, Gib10/02 or Vector: 5㎕

Both insert and control are cultured in each Petri plate.
The Petri plates will be checked tomorrow.

2022/10/05

Kazuma Tsurumaki

  1. Checked the Petri plates Colonies were observed.

  2. Culturing Acid-producing bacteria

    LB mediums: 3ml, IPTG: 3μl, Culture fluid for acetic acid-producing strains: 30μl

    Cultured overnight

2022/10/06

Kazuma Tsurumaki

  1. Made Glycerol stock of acid-producing bacteria mixed 500㎕ of culture fluid cultured from yesterday for acetic acid-producing strains and 20% glycerol. frozen at -80℃. named the products “aatA-poxB”.

2022/10/08

Kazuma Tsurumaki, Kota Toyoda

  1. Made LB medium Since the LB medium made last month was muddy, we determined that this was contaminated. Therefore, we made new mediums. Both ager and liquid medium are 200ml.

  2. Miniprep cellulose-degrading bacteria Following Promega's protocol, minipreped samples of cellulose-degrading strain picked at 20221005. named the product “glc-plasmid10/8”.

  3. Restriction Enzyme Treatment of glc-plasmid10/8 For Agarose gel electrophoresis, cut glc-plasmid10/8 with EcoRI, according to the protocol offered by New England Bio Labs

  4. Gibson Assembly

glu-ace2: 5㎕, glue: 0.5㎕, vector: 1.5㎕, milliQ:3㎕, master mix(2x):10㎕

  1. Transformation of aatA-poxB Culture fluid: 100㎕ Both insert and control are cultured in each Petri plate

  2. Dissolved cellulose

Procedure

Dispense LB medium, cellulose powder, culture fluid, and ampicillin (100x) into each culture tube. Culture in shaking water bass at 37℃ for 5 hours or more.

Made 3 samples. Each tube contains

> LB medium: 6ml
> Cellulose: 0.06g
> Culture fluid: 30μl
> Ampicillin: 6μl

Cultured overnight from 17:00, 20221008.

2022/10/09

Kazuma Tsurumaki, Kota Toyoda

  1. Agarose gel electrophoresis Ran gel electrophoresis to determine whether glc-plasmid10/8 was as designed. Used glc-plasmid cut at 20221008 with EcoRI.

  2. Dissolved cellulose

    Each tube contains: Cellulose: 0.03g, Ampicillin(100x): 0.3μl, LB medium:3ml, Culture fluid : 3μl

    Three controls and three inserts each were prepared. Cultured overnight in a shaking water bath.

2022/10/10

Kazuma Tsurumaki, Kota Toyoda

  1. Dissolved cellulose

    Glucose assay protocol: According to the protocol offered by the manufacturer.
    https://www.dojindo.co.jp/products/G264/img/g264_manual.pdf
    To remove particles, centrifuge 80μL of samples at 10000rpm, 27℃ for 1min and use 50μL of supernatant fluid.

  2. Determine the concentration of glucose by assay kit

    11:45 Sampling 12:15 Mix working reagent and samples 12:45 Measure absorbance

    Abosorbrances are much lower than only LB

    13:02 Mix working reagent and samples which are not centrifuged

    Desided not to centrifuge samples for that time.

    Result: (mM)

    The transformed E.coli will have been cultured overnight.

2022/10/11

Kota Toyoda

  1. Determine the concentration of glucose by assay kit at 9:15 Result(mM);


    Much lower than yesterday

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