Team:SJTU-BioX-Shanghai/Protocol

Protocol

Section1

  • Western Blotting protocol

    1、Prepare of Proteins
    1.1 Culture E.coli BL21(DE3) with LB(l) medium overnight at 37℃, 200rpm
    1.2 Add 1ml overnight culture into 5ml new LB(l) medium for 1 hour at 37℃, 200rpm
    1.3 Induce culture by IPTG (with final concentration of 0.5mM) more than 4 hours, while the OD600 is 0.6
    1.4 (Lysis) Add 1.5ml lysozyme (1mg/ml) into 2ml centrifuged and the supernatant discadred strain, 30℃ for 30 minutes
    1.5 (Degeneration) Add SDS loading buffer and put the centrifugal tube into boiled water for 10 minutes
    2、Prepare SDS-PAGE gel, Running buffer, Trans buffer and TBST Washing buffer
    SDS-PAGE:
    12% Separation Gel(the lower gel):
    30%Acr-Bis      3.2ml
    1.5M Tris-HCl(pH=8.8) 2ml
    10%SDS         80μl
    10%AP          80μl
    TEMED         3μl
    H2O         2.64ml
    Total         8ml
    5% Spacer Gel(the upper gel):
    30%Acr-Bis      500μl
    1.5M Tris-HCl(pH=6.8) 500μl
    10%SDS         40μl
    10%AP         30μl
    TEMED         3μl
    H2O         2ml
    Total         3ml
    1×Running buffer:
    Tris      3.03g
    Glycine    14.4g
    10%SDS    10ml
    Add ultra pure water to 1L
    Store at 4℃
    1×Trans buffer:
    Tris      3.03g
    Glycine    14.4g
    10%SDS     10ml
    Methanol    200ml
    Add ultra pure water to 1L
    Store at 4℃
    1×TBS buffer:
    Tris      2.4g
    NaCl     8.7g
    Adjust pH=7.6 with HCl
    Add ultra pure water to 1L
    TBST Washing buffer:
    TBS        500ml
    Tween-20     250μl
    Blocking buffer:
    Add 2.5g no-fat dry milk into 50ml TBST Washing buffer
    Prepare Blocking buffer only when using it
    3、Protein transfer and Immunodetection
    3.1 Add 15μl sample and 5μl Protein marker
    3.2 Add Running buffer for electrophoresis with a constant voltage at 100V for about 25 minutes, then change voltage into 200V for 40 minutes (adjust the former time by the marker)
    3.3 Open the package and cut the upper spacer gel
    3.4 Prepare the same size PVDF membrane and put into Trans buffer for 5 minutes before trans-membrane
    3.5 Open gel cassettes and put the gel on the PVDF membrane sandwiched between two pieces of blotting paper(always filter paper)
    Mind the order of gel and PVDF membrane between the electrodes, black end with gel for negative electrode and white end with PVDF membrane for positive electrode.
    3.6 Add Trans buffer for electrophoresis with a constant current at 350mA for 65 minutes(Crowd with ice at a 4℃ temperature )
    3.7 After trans-membrane, wash PVDF membrane with 10ml TBST for 10 minutes with gentle shaking
    3.8 Add 10ml Blocking buffer with gentle shaking for 1 hour at room temperature or 4℃ overnight
    3.9 Wash PVDF membrane with 10ml TBST for 10 minutes with gentle shaking
    3.10 Add 10ml diluted(1:5000) primary antibody for 2 hours at room temperature or 4℃ overnight with gentle shaking (primary antibody can be used repeatedly for 10 times)
    3.11 repeat step 3.9
    3.12 Add 10ml diluted secondary conjugated antibody for 2 hours at room temperature or 4℃ overnight with gentle shaking (seconfary antibody needn’t be repeatedly used)
    3.13 repeat step 3.9
    3.14 With the ratio of 1:1 to mix Luminous substrate A and B, typically 200-400μl mixture added on PVDF membrane
    3.15 Take photos by specific machine and store it

    • Instructions: you have the change not only the main text, but also have to modify the text in the navgation bar on the left side. Now put your content here and do the same for the following sections. xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx Note template is here --- OD OD Optical density .

    Mouse experiments

    • CRC induction

      Subject: C57BL/6 mice strain (6~8 week old)
      Material: Azoxymethane (AOM) solution (1mg/ml)
          Dextran Sulfate Sodium Salt (DSS) solution (2.0% & 2.5%)
      Experiment aim:
        After analyzing the qualities of our device that requires testifying meanwhile thoroughly considering the 3Rs principle, we have reduced the need of animal experiments to the least, but for certain qualities the testifying procedure is difficult to replace, refine and reduce experimental animals use. As a result the colorectal cancer mouse model was induced in the minimum amount just to be adequate for product testifying procedure.
      Experiment design:
        Azoxymethane (AOM) is a chemical agent that can initiate cancer by alkylation of DNA, there by facilitating base mispairings, while Dextran Sulfate Sodium Salt (DSS) is a substance that causes inflammations that will eventually lead to enteritidis.
        After being injected into the subject, AOM introduces a carcinogenic environment in vivo, along with DSS consumed, enteritidis is likely to occur and soon develop into colorectal cancer.
      Experimental procedure:
        After the mice strain stabilizes and being weighed, intraperitoneal injection of AOM solution is given in the amount of 10mg/kg and let rest for a week.
        With the AOM injection completed, the DSS induction process begins. One induction cycle lasts for 3 weeks while the complete induction process includes 3 cycles of which. In the first week of the three-week cycle the DSS solution (2.0% & 2.5%) was given as drinking water to our subject. DSS solution was mixed right before it was given to subject to ensure the best results. After one week of consuming DSS, the drinking water for the next two weeks was switched to normal clear water. Repeat the cycle two more times while carefully monitor the weight change of the subject.
      Citation:
      [1]Ameet I. Thaker, Anisa Shaker, M. Suprada Rao, Matthew A. Ciorba,Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS) ,Journal of Visualized Experiments,9/11/2012
      [2]Clemens Neufert, Christoph Becker & Markus F Neurath,An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression,NatureProtocol, 9/08/2007

    • Structure observation of CRC models—paraffin sectioning and HE staining

      Subject: Colorectal tissue from CRC induced mouse model and normal ones
      Material: PBS, different concentration of ethanol (50%, 75%, 85%, 95%, 100%), xylene, paraffin wax, haematoxylin and eosin (HE stain)
      Experiment aim:
        Before using our CRC mouse model for any further experiment, it is essential to first testify the existence of the disease. The exquisite imaging of the cancerous area is also critical in modeling as well as device improvement.
      Experiment design:
        To maintain the original structure of the tissue, the paraffin sectioning was carry out. After receiving a clear and complete biopsy of the sample, haematoxylin and eosin (HE) stain was performed to get the best vision of the histological structure.
      Experiment procedure:
        First, a section of colon or rectum was removed from the subjects and longitudinally cut open to form a rectangular piece of tissue. The tissue was then placed on a small cardboard and soaked in paraformaldehyde (PFA) solution overnight. Then the tissue was washed with phosphate buffered saline solution (PBS) three times (10minutes each time). With the tissue cleared from PFA, a series of soaking process was done on decoloring shaker. The soaking process was shown in table below for better understanding.(table here)
        After the soaking process the samples were then embedded in paraffin wax and sliced into biopsy of 10μm thick. They were gently flushed with PBS before the haematoxylin was applied and let sit in the dark for 4 minutes. Haematoxylin was then removed and the samples were rinsed and flushed in ddH2O for 5 minutes. Add 4 drops of HCl-Ethanol (1ml HCl+400ml ethanol) and rapidly dip the sample in ddH2O where a 2-minute flush as well as a 2-minute soaking were performed. Eosin was applied and last on the biopsy for 15 seconds before it was flushed out gently using ddH2O. Similar to the paraffin sectioning, a series of soaking process on decoloring shaker was involved and mentioned in table below. The biopsy was let dry in room temperature and sealed with neutral balsam to complete the HE stained slide.(table here)

    • Examination of T antigen expression level

      The examination involved two examining method: the cellular level examination and the biopsy examination.
      Subject: Colorectal tissue from CRC induced mouse model and normal ones
      Material: Trypsin, PNA-FITC, PBS, ddH2O
      Experiment aim:
        Through document material we knew that the T antigen expression level of cancerous area is much higher than normal tissue, and this characteristic plays an important role in our project design. So before we started to testify our product’s quality, it is essential to solidify the foundation of our product design. Experiment design:
        In this experiment we use the fluorescent level of PNA-FITC to indicate the expression of T antigen. PNA-FITC is a plant lectin protein that binds with the sugar that contains carbohydrate sequence Gal-β(1-3)-GalNAc, a sequence also carries by our testifying target, T antigen. Also, the frozen section of the tissue makes the binding with cellular dye better to observe. After adequate binding time being given, we can compare the fluorescent level of CRC mouse model with normal ones to reach to our conclusion.
      Experimental procedure:
      C.1 cellular level examination
        First, the colorectal tissue was removed from the subjects and digested by trypsin under 37°C for 15~20 minutes. The volume of the trypsin used is four times of the sample with the concentration of 0.25%. The liquid should then be centrifuged for 10 minutes in 1000XG and remove the supernatant to eliminate further damage by trypsin remained. Phosphate buffered saline (PBS) was added to the remains before the samples were resuspended. The mixture were then filtered through 50μm filter membrane and transport to new lightproof sample tubes. A drop of the sample was drawn and observed under microscope to ensure the density of which is around 106cell/ml. If the cell number is lower than target, the samples should be centrifuged again and withdraw certain amount of the supernatant. While in higher cell count situation, extra PBS should be added to dilute the sample to the ultimate density.
        Then PNA-FITC(1μg/ml) was added to the samples and was incubated under 37°C for 30 minutes. Centrifuge the samples for 10 minutes in 1000XG and replace the supernatant with clear PBS to eliminate the possible interrupting signals caused by unbunded PNA-FITC. Our samples were then measured using flow cytometry. When using the cytometry we circled out the living cells on the graph and compare the fluorescent level chart between CRC models and normal mouse.
      C.2 Biopsy examination
        The tissue was fixed by frozen section before we apply PNA-FITC on to the biopsy. So first, a section of colon or rectum was removed from the subjects and longitudinally cut open to form a rectangular piece of tissue. The tissue was then placed on a small cardboard and soaked in paraformaldehyde (PFA) solution overnight. The soaking solution was then switched to 30% sucrose solution and last for another night. Put the tissue in freezing solution and freeze under -20°C overnight to prepare the following biopsy. The properly frozen tissue were sliced into biopsy of 10μm thick.
        In the following procedure we refer PNA-FITC to as the primary antibody and carry out the standard procedure of immunohistochemical test. The biopsy was first flushed with 0.1% tween-20 phosphate buffered saline solution (PBST) three times (10minutes each time), and then the blocking liquid (5% FBS+0.1% Triton X-100) was added with the incubation last for an hour. After removing the blocking liquid the samples require another three-time flushing by PBST. At last PNA-FITC (50μg/ml) was added and the samples were incubated in 4°C overnight. Before being observed under fluorescent microscope, a three-time PBST flushing was done again. The final samples were sealed with mounting medium that contains 4′,6-diamidino-2-phenylindole (DAPI) for better vision on the structure as well.

    • Bacterial adhesion property examination

      Subject: Colorectal tissue from CRC induced mouse model and normal ones Material: Two types of engineered bacteria: A. bacteria with surface peptide
                            expression and GFP
                          B. bacteria with only GFP
           PBS
      Experiment aim:
        After solidifying the distinguishing T antigen expression level in experiment B as well as the adhesion property in cellular level, it is time to apply our product directly on tissue to determine the adhesion property in real situation.
      Experimental design:
        Since the overall examination of fluorescent level of the tissue is difficult to perform, we’ve decided to apply our device straight on the tissue before performing a frozen section. Frozen section comparing to paraffin section enables the tissue to maintain the binding with bacteria better so it was performed soon after the bacterial adhesion procedure.
      Experimental procedure:
        First, a section of colon or rectum was removed from the subjects and longitudinally cut open to form a rectangular piece of tissue. The tissue were then placed on a small cardboard and soaked in paraformaldehyde (PFA) solution overnight. The soaking solution was then switched to sucrose solution (40%) and last for another night. Meanwhile our engineered bacteria were incubated and induced until the final OD600=1 with obvious GFP observed under fluorescent microscope.
        Then, the tissue were separately put in the two kind of bacteria media and were incubated under 37°C for 30 minutes. After the incubation the tissue were soaked in PBS and gently washed for three times using PBS. Put the tissue in freezing solution and freeze under -20°C overnight to prepare for the following biopsy. The properly frozen tissue were slice into pieces of 10μm thick to form a biopsy. The biopsy was then observed under fluorescent microscope to compare the aggregation of the glowing bacteria.

    • Your title3

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    Section3

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    2018 Interlab Plate Reader Protocol
    Protocols/Transformation

    • Western Blotting protocol

      1、Prepare of Proteins
      1.1 Culture E.coli BL21(DE3) with LB(l) medium overnight at 37℃, 200rpm
      1.2 Add 1ml overnight culture into 5ml new LB(l) medium for 1 hour at 37℃, 200rpm
      1.3 Induce culture by IPTG (with final concentration of 0.5mM) more than 4 hours, while the OD600 is 0.6
      1.4 (Lysis) Add 1.5ml lysozyme (1mg/ml) into 2ml centrifuged and the supernatant discadred strain, 30℃ for 30 minutes
      1.5 (Degeneration) Add SDS loading buffer and put the centrifugal tube into boiled water for 10 minutes
      2、Prepare SDS-PAGE gel, Running buffer, Trans buffer and TBST Washing buffer
      SDS-PAGE:
      12% Separation Gel(the lower gel):
      30%Acr-Bis      3.2ml
      1.5M Tris-HCl(pH=8.8) 2ml
      10%SDS         80μl
      10%AP          80μl
      TEMED         3μl
      H2O         2.64ml
      Total         8ml
      5% Spacer Gel(the upper gel):
      30%Acr-Bis      500μl
      1.5M Tris-HCl(pH=6.8) 500μl
      10%SDS         40μl
      10%AP         30μl
      TEMED         3μl
      H2O         2ml
      Total         3ml
      1×Running buffer:
      Tris      3.03g
      Glycine    14.4g
      10%SDS    10ml
      Add ultra pure water to 1L
      Store at 4℃
      1×Trans buffer:
      Tris      3.03g
      Glycine    14.4g
      10%SDS     10ml
      Methanol    200ml
      Add ultra pure water to 1L
      Store at 4℃
      1×TBS buffer:
      Tris      2.4g
      NaCl     8.7g
      Adjust pH=7.6 with HCl
      Add ultra pure water to 1L
      TBST Washing buffer:
      TBS        500ml
      Tween-20     250μl
      Blocking buffer:
      Add 2.5g no-fat dry milk into 50ml TBST Washing buffer
      Prepare Blocking buffer only when using it
      3、Protein transfer and Immunodetection
      3.1 Add 15μl sample and 5μl Protein marker
      3.2 Add Running buffer for electrophoresis with a constant voltage at 100V for about 25 minutes, then change voltage into 200V for 40 minutes (adjust the former time by the marker)
      3.3 Open the package and cut the upper spacer gel
      3.4 Prepare the same size PVDF membrane and put into Trans buffer for 5 minutes before trans-membrane
      3.5 Open gel cassettes and put the gel on the PVDF membrane sandwiched between two pieces of blotting paper(always filter paper)
      Mind the order of gel and PVDF membrane between the electrodes, black end with gel for negative electrode and white end with PVDF membrane for positive electrode.
      3.6 Add Trans buffer for electrophoresis with a constant current at 350mA for 65 minutes(Crowd with ice at a 4℃ temperature )
      3.7 After trans-membrane, wash PVDF membrane with 10ml TBST for 10 minutes with gentle shaking
      3.8 Add 10ml Blocking buffer with gentle shaking for 1 hour at room temperature or 4℃ overnight
      3.9 Wash PVDF membrane with 10ml TBST for 10 minutes with gentle shaking
      3.10 Add 10ml diluted(1:5000) primary antibody for 2 hours at room temperature or 4℃ overnight with gentle shaking (primary antibody can be used repeatedly for 10 times)
      3.11 repeat step 3.9
      3.12 Add 10ml diluted secondary conjugated antibody for 2 hours at room temperature or 4℃ overnight with gentle shaking (seconfary antibody needn’t be repeatedly used)
      3.13 repeat step 3.9
      3.14 With the ratio of 1:1 to mix Luminous substrate A and B, typically 200-400μl mixture added on PVDF membrane
      3.15 Take photos by specific machine and store it

      • Instructions: you have the change not only the main text, but also have to modify the text in the navgation bar on the left side. Now put your content here and do the same for the following sections. xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx Note template is here --- OD OD Optical density .

      Section

      xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxxxxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx

      • Your title1

        xxx xx xxxx xxx xxx xxx xx xxxx xxx xxxxx xxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxx

      • Your title2

        xxx xx xxxx xxx xxx xxx xx xxxx xxx xxxxx xxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxx

      • Your title3

        xxx xx xxxx xxx xxx xxx xx xxxx xxx xxxxx xxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxxx xx xxxx xxx xxxxxxx

      Section3

      xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx. The text-link template is here.

      2018 Interlab Plate Reader Protocol
      Protocols/Transformation

      section4

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      Fig 1. The particle standard curve obtained form the 2nd calibration experiment.

      section4

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    Fig 1. The particle standard curve obtained form the 2nd calibration experiment.

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    The table template is here.

    Table 1. Colony forming units per 0.1 OD600

    samples dilution factor CFU/mL
    8×104 8×105 8×106
    1.1 TNTC 48 11 3.84E+07
    1.2 248 41 10 3.28E+07
    1.3 172 54 5 4.32E+07
    2.1 TNTC 143 20 1.14E+08
    2.2 TNTC 153 25 1.22E+08
    2.3 TNTC 151 18 1.21E+08
    3.1 TNTC 119 16 9.52E+07
    3.2 TNTC 125 19 1.00E+08
    3.3 TNTC 89 18 7.12E+07
    4.1 TNTC 209 16 1.67E+08
    4.2 TNTC 130 17 1.04E+08
    4.3 TNTC 164 10 1.31E+08

    US imaging

    We use the Fujifilm VisualSonics / VEVO LAZR-X imaging system and MX250 transducer. The application is set to VA Phantom. Parameters are listed below:

    • Frequency: 21MHz
    • Power for imaging: min(2%)
    • Power for collapse: max(100%) (sustaining for 5 minutes)
    • Max frame: 50
    • Gain: 16dB
    • Focus: adjust to the depth of the layer where engineered E.coli lies in.
    • Field of view: adjust to achieve the maximum repression of noise and also zoom in on the interest region as much as possible.

    Preparation before imaging: PBS solution is needed to cover the imaging samples allowing the transducer to dip into the solution. Pay attention to not producing any unwanted bubbles.