Protocols for Biosensor Construction

From BioDesign for the Real-World
Jump to: navigation, search

These protocols were prepared by the van der Meer lab at UNIL for the BRAAVOO Workshop and Creative Design Course / Biodesign for the Real World Winter School 2016.

Cloning into Plasmid Vectors:

Preparation of DNA for traditional cloning methods is dependent upon restriction enzyme digestion to generate compatible ends capable of being ligated together. In this course, the DNA to be cloned is a previously cloned gene that needs to be moved from one vector to another (subcloning). Subcloning requires the use of restriction enzymes that cut immediately outside the insert fragment without cutting within the insert itself. Two restriction enzymes are used to digest the DNA resulting in directional insertion into the compatible plasmid. Restriction enzymes that have a recognition site within the multiple cloning site (MCS) are commonly used since they do not cut elsewhere in the vector DNA and typically produce two easily resolved DNA fragments.

Cloning Protocols:

Plasmid DNA purification (done with JETSTAR Plasmid Purification Kit):

Culture Volumes and DNA Yields
GENOMED recommends LB medium to grow E. coli cells to prepare plasmid DNA with JETSTAR columns. The cell density should be ≈ 1 x 109 cells per ml medium (1-1.5 A600 units/ml)

Culture Volume (for Midi) DNA Yield

High Copy Plasmids 15 - 25 ml, 45 - 100 μg
Low Copy Plasmids 25 - 100 ml, 5 - 100 μg

Protocol (for Midi)

1. Column Equilibration
Before the cleared lysate is prepared, columns are equilibrated by applying 10 ml solution E4
Allow the column to empty by gravity flow. Do not force out remaining solution.
2. Harvesting Bacterial Cells
E. coli cells are pelleted by centrifugation. Remove all traces of medium
3. Cell Resuspending
Add 4 ml solution E1 to the pellet and resuspend the cells until the suspension is homogeneous
4. Cell Lysis
Add 4 ml solution E2 and mix gently but thoroughly by inverting until the lysate appears to be homogeneous. Do not vortex! Incubate at room temp. for 5 min
5. Neutralization
Add 4 ml solution E3 and mix immediately by multiple inverting until a homogeneous suspension is obtained. Do not vortex!
Centrifuge the mixture at room temperature and 12.000 x g for 10 min
6. Column Loading
Apply the supernatant from step 5 to the equilibrated JETSTAR column
Allow the lysate to run by gravity flow
7. Column Washing
Wash the column with 10 ml solution E5 twice. Allow the column for each wash to empty by gravity flow
8. Plasmid Elution
Elute the DNA with 5 ml solution E6. Do not force out remaining solution
9. Plasmid Precipitation
Precipitate the DNA with 0.7 volumes of isopropanol (3.5 ml). Centrifuge at 4°C and ’000 x g for 30 min
Wash the plasmid DNA with 70% ethanol and re-centrifuge
Air dry the pellet for 10 min, and re-dissolve the DNA in a suitable volume of buffer or water


Restriction Enzyme digestions:

Restriction Enzyme digestion

Materials required

-Restriction Enzymes (New England Biolabs)
-NEBuffer 10x
-Water bath at 37°C

Procedure

1. Insert double digestion
I + E1 & E2
H2O to 100 µl
10X Buffer 10 µl 1X Buffer
Midiprep (Insert) 5-15 µl 3 µg
Enzyme1 2 µl 40U
Enzyme2 2 µl 40U
I = plasmid containing Insert DNA
  • Cut the plasmid containing the insert with two restriction enzymes
  • Mix well by pipetting
  • Incubate at 37°C for 2 h


2. Vector plasmid single digestions
V + E1 V + E2
H2O to 100 µl
10X Buffer 10 µl 1X Buffer 1X Buffer
Midiprep (Vector) 5-15 µl 3 µg 3 µg
Enzyme1 2 µl 40 U
Enzyme2 2 µl 40U
V = Vector (pPROBE-egfp)
  • Cut the plasmid vector with each of the two enzymes as for isolating the Insert
  • Mix well by pipetting
  • Transfer 20 µl of each single digestion mixture into a new (labelled) Eppendorf tube to use as control for each enzymatic digestion
  • Pool the two single digestions (2x 80 µl) to end up with a double digestion
  • Incubate at 37°C for 2 h


3. Vector Dephosphorylation
Dephosphorylation reduces background in cloning, preventing empty vectors from forming. (This website says this in another way, and here is a picture of what happens) that allows you to increase the likelihood of getting your inset cloned!
TSAP Thermosensitive Alkaline Phosphatase catalyzes the removal of 5´ phosphate groups from DNA, thus preventing the recircularization and religation of linearized cloning vector DNA during ligation
  • After enzymatic digestion (or after 1h30), add 3 µl TSAP ONLY TO DIGESTED VECTOR and let the reaction run 15-30 min, at 37°C


Agarose gel electrophoresis:

Materials required

-Tris-acetate buffer (TAE) 1x
-Agarose
-SybrSafe solution (10’000x) !!! potentially mutagenic
-Ethidium bromide solution 10 mg/ml (Alternative to SybrSafe) !!! highly carcinogenic
-Gel loading dye (6x)
Pre-mixed loading buffer used to prepare samples for loading on agarose gels. It contains one dye for visual tracking of DNA migration during electrophoresis. Bromophenol blue is the standard tracking dye for electrophoresis. The presence of glycerol ensures that the DNA in the sample forms a layer at the bottom of the well
-DNA size standard (Mass Ruler DNA Ladder Mix, Thermo Fisher Scientific)

Preparation of agarose gel 1%

This gel is thick, for gel extraction

Mix 0.5 g (0.75 g) agarose and 50 ml (75 ml) TAE 1x
Melt this mixture completely in the microwave oven, stir to mix
Let cool down to 60°C
Add 10 µl SybrSafe (or 4 µl ethidium bromide)
  • Pour the gel into the casting tray and insert the comb
  • Allow the gel to solidify for at least 20 min
  • Put gel into electrophoresis tank and fill the latter with TAE 1x buffer just to cover the gel
  • Carefully remove the comb

Gel loading and electrophoresis

  • Briefly centrifuge your samples
  • Add loading dye (6x) to your samples to a 1x final concentration:
  • Insert double digestion: 100 µl digestion reaction + 20 µl loading dye 6x
  • Vector double digestion: 160 µl digestion reaction + 30 µl loading dye 6x
  • Vector control digestion E1: 20 µl single digestion reaction control + 4 µl loading dye 6x
  • Vector control digestion E2: 20 µl single digestion reaction control + 4 µl loading dye 6x
  • Mix by pipetting several times up and down, then load the samples into the gel slots (wells):
1 & 2. Load 60 µl of Insert digestion reaction into the two first wells
3 to 5. Load 60 µl of Vector digestion reaction into the three next wells
6. Load 8 µl of a DNA standard into the 6th slot
7. Load 24 µl of Vector control digestion E1 into the next well
8. Load 24 µl of Vector control digestion E2 into the next well
  • Run the gel at 100 V for 30 to 60 min (depending on the size of the DNA fragments)
  • Take a picture of your gel on the trans-illuminator and calculate the sizes of your DNA fragments by using the DNA standard as a reference
Note: expose DNA to UV shortly to avoid mutations!!!


Purification of DNA fragments from agarose gel:

_Isolate the Insert DNA band (Insert E1/E2)
_Isolate the vector fragment (Vector E1/E2)

Materials required

-Nucleospin columns
These are based upon SiO2 (silicium dioxide) as the main component, and you can look here for more info on the purification process.
-Buffers NTI, NT3 and NE
-Thermoblock or water-bath set to 50°C

DNA purification from the agarose gel

Here, we use the “Nucleospin Gel and PCR clean-up kit”, Macherey Nagel

  • Cut DNA band from the gel using sharp scalpel
  • Minimize gel volume by cutting the excess if needed
  • Transfer to a pre-weighed Eppendorf tube
  • Weigh the agarose block
  • Add 200 µl of NTI per 100 mg of gel (scale if necessary but keep the volume : weigh ratio to 2:1)
  • Incubate at 50°C for 5-10 min until gel is dissolved (Vortex twice during incubation)
  • Place the column in a provided 2 ml collection tube in a suitable rack
  • Apply the mixture of DNA-NTI buffer to the column and centrifuge 1 min at 13’000 rpm
  • Discard the flow-through, blot off the liquid and place column back in the same tube
  • Add 0.7 ml NT3 buffer and centrifuge 1 min at 13’000 rpm
  • Discard the flow-through, blot off the liquid and place column back in the same tube
  • Repeat washing:
  • Add 0.7 ml NT3 buffer and centrifuge 1 min at 13’000 rpm
  • Discard the flow-through, blot off the liquid and place column back in the same tube
  • Dry column by centrifugation: 2 min at 13’000 rpm to remove residual NT3 buffer
  • Place column into a clean Eppendorf tube
  • Elute DNA:
  • Add 12 µl of water to the center of the column
  • Let column stand for 1 min, then centrifuge 1 min at 8’000 rpm
  • Discard the column but Keep the flow-through!!! It contains your purified DNA product

NanoDrop quantification

This step is after purification of DNA fragments from agarose gel

  • Insert E1/E2 = 21 ng
  • Vector E1/E2 = 18.6 ng

    Ligation of Plasmid Vector and Insert DNA:

    Ligation

    Clone the Insert E1/E2 into the Vector E1/E2

    1. Vector:Insert ratio
    After the vector and insert DNA have been prepared for ligation and the concentration of each estimated (using Nanodrop), calculate the insert quantity for Vector:Insert ratio of 1:5
    Insert quantity (ng) =
    ng vector x Kb size of insert x 5 Kb size of vector 1
    Example of Ligation Mix (using LigaFastTM Rapid DNA Ligation System)

    :Control Ligation

    H2O (to 10 µl) 1µl 0µl
    2X Rapid Ligation Buffer 5µl 5µl
    Vector pPR E1/E2 3µl 3µl
    Insert E1/E2 - 1µl
    T4 DNA Ligase 1µl 1µl
    Incubate ligation reaction at 25°C for 1-2h
    Use 0.2 ml tubes known to have low DNA-binding capacity
    The LigaFastTM Rapid DNA Ligation System is designed for the efficient ligation of cohesive-ended DNA inserts into plasmid vectors in just 15 minutes


    Transformation of DNA into Bacteria:

    Transformation into E. coli DH5a or/and MG1655

    Materials required

    -200 µl aliquots of competent cells
    -Water-Bath at 42°C
    -Ice
    -SOC medium
    -Selection plates (LB-agar plus antibiotics)

    Heat shock Transformation of Competent Cells

    • Thaw a 200 µl aliquot of competent cells on ice for15 min
    • Transfer 100 µl of competent cells into a new ice cold Eppendorf tube (labelled C, for ligation control)
    • Add 5 µl of ligation mixture to competent cells and mix by gently swirling the pipet tip – do not mix by pipetting
    • Add 5 µl of ligation control mixture to competent cells and mix by gently swirling the pipet tip
    • Incubate on ice for 15-30 min
    Note: The volume of DNA solution added to competent cells must not exceed 10µl. Optimal transformation efficiencies are observed with up to 10 ng of DNA; larger quantities may cause a decrease in efficiency with some competent cell preparations
    • Heat the tube at 42°C (in a waterbath) for 90 sec
    • Immediately, place the tube on ice to cool for 2 min
    • Add 0.9 ml of SOC medium and incubate the cell suspension for 1 h at 37°C, with shaking at 180 rpm
    • Plate the transformation mix onto 2 selection plates (LB + Km50):
    • 100 µl cell suspension on the 1st plate,
    • ≈ 400 µl on the 2nd plate


    DNA amplification by PCR (for analysis of transformants):

    PCR

    Recombinants obtained from cloning are screened by PCR to identify the clone of interest

    Materials Required

    PCR mix, vol. 50µl (example for 12 PCRs, 9 transformants + 2 controls)
    Products Volume Comments
    H2O 441 µl ad 600 µl
    Go Taq Buffer 5X 120 µl 1/5 final volume
    dNTP 10 mM 12 µl
    Primer fwd 12 µl In Insert
    Primer rev 12 µl In Vector
    DNA Template 0 µl Transformants (colonies)
    GoTaq DNA polymerase 3 µl Keep on ICE, add at the end

    PCR program:

    5 min 94ºC 1 Initial denaturation
    45 sec 94ºC 30 Denaturation
    30 sec 55ºC Hybridization, Tº depends on primers sequence (0 à 2°C < "lower" T°)
    30 sec 72ºC Elongation: depends on the number of pb to amplify (1 min per Kb)
    7 min 72ºC 1
    Infinite 4ºC 1 15°C if overnight PCR


    Related Documentation

    • Detailed Cloning Protocols for Particular Biosensor Constructs made by course participants

    Octane (AlkST-GFP)

    AlkST-GFP cloning protocol

    Arsenic (ArsR-GFP)

    ArsR-GFP cloning protocol

    Mercury (MerR)

    MerR-GFP cloning protocol

    Zinc (ZntA)

    ZntA-GFP cloning protocol