Team:Tsinghua/Experiments
Experiments / Protocols
Protein expression in E. coli
We exploited E. coli BL21(DE3) cells to transiently express protein elements. The proteins of interest are cloned into IPTG-inducible systems under distinct promoters.
1. Inoculate a single colony in 3 mL LB medium, incubate at 37℃, 220 rpm overnight.
2. Inoculate 1 mL bacteria into 50 mL LB medium in bottle, incubate at 37 ℃, 220 rpm for about 2~5 h until O.D.600 reaches 0.6~1.0.
3. Add IPTG of final concentrations 10-3 to 2 mM according to different assays.
4. Express protein at 37℃, 220rpm for 2h or at 16℃, 220 rpm for 10h.
Microscopy
We used NIKON A1 microscope with 100 × oil immersion lens for imaging. And the microscopic slides of bacteria samples were made for confocal imaging.
Fluorescence recovery after photobleaching (FRAP).
We photobleached green fluorescence puncta within E. coli with a 488 nm laser pulse (10%-20% intensity; dwell time 0.125-0.5 s). And we record the intensity change with time.
Light induction.
For light induction, we used a 488 nm laser pulse (2%-3% intensity; dwell time 8 s) to stimulate a specific region. We then recorded its change for 10-15 min after stimulation.
3D imaging.
For 3D imaging, the sample was scanned at every 10 μm focal plane within a range of 100 μm. Then the images of different focal planes were integrated by the software and a 3D image of the sample was generated.
Rluc
Materials
› LB culture
› Coelenterazine (clz)
› dissolved in ethanol (10mM) or stay freeze-dried and store in -80C, heat before using if there is precipitate.
› Spontanously oxidize anyway.
› PBS
Rluc
Procedure
1. Coelenterazine (clz, from Invitrogen) dissolved in ethanol (10mM) or stay freeze-dried and store in -80C, heat before using if there is precipitate.
2. Bacteria grown to an OD 0.6-0.8, and add IPTG. Shake for 4 h at 22℃ on a rotary shaker to an OD~1. The culture was washed twice with PBS (pH 6, slightly acidic to avoid oxidization of clz), adjusted to an OD of 0.4–0.5, and aliquoted 100ul/well into a black 96-well plate in triplicate (or 2ml in a cuvette).
3. Prior to each assay, a single aliquot of clz was thawed, diluted in PBS to a final concentration of 1 mM, and incubated in the dark at room temperature for a few minutes.
4. Clz was added to a final concentration of 30 μM to the PBS-washed cells, which were subsequently incubated in the dark for 30 min at room temperature to allow the luciferase-generated signal to stabilize (stir to make bubbles if in cuvette).
5. The total luminescence of each well was then measured every minute for a total of 5 min with an integration time of 1 sec using a Microplate Reader.
Rluc
Microplate Reader measurement
1. Incubate samples to reach 37C.
2. First OD600 of the bacteria is measured to determine the amount.
3. Intensity of florescence is measured to see if Rluc is expressed.
4. Luminance of sample is measured 3 times to determine the background values of the samples.
5. Add substrates according to protocol.
6. Luminance of sample is measured 10 times to determine velocity of the reaction.
Rluc
Data analysis
Average all luminance value(after addition of clz) and normalize the average to OD600 of the sample.
1.Set up the following reaction on ice:
| Recommended Amount of Fragments Used for Assembly | |||
| 2–3 Fragment Assembly* | 4–6 Fragment Assembly** | Positive Control✝ | |
| Recommended DNA Molar Ratio | vector:insert = 1:2 | vector:insert = 1:1 | |
| Total Amount of Fragments | 0.03–0.2 pmols* | 0.2–0.5 pmols** | 10 μl |
| X μl | X μl | ||
| NEBuilder HiFi DNA Assembly Master Mix |
10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μl✝✝ | 20 μl✝✝ | 20 μl |
| * | Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. | ||
| ** | To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each). | ||
| † | Control reagents are provided for 5 experiments. | ||
| †† | If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix. | ||
2.Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.
Reaction setup:We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.
| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
| Q5 High-Fidelity 2X Master Mix | 12.5 µl | 25 µl | 1X |
| 10 µM Forward Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 1,000 ng |
| Nuclease-Free Water | to 25 µl | to 50 µl |
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Transfer PCR tubes to a PCR machine and begin thermocycling.
Thermocycling Conditions for a Routine PCR:
| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 25–35 Cycles | 98°C | 5–10 seconds |
| *50–72°C | 10–30 seconds | |
| 72°C | 20–30 seconds/kb | |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C |
*It depends on the sequence of primers.
General Guidelines for templates:
Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:
| DNA | AMOUNT |
| DNA Genomic | 1 ng–1 µg |
| Plasmid or Viral | 1 pg–10 ng |
General Guidelines for primers:
Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.
General reaction mixture for PCR (total 50 μl)
Premix Taq Hot Start Version＊ 25 μl
Template< 500ng
Primer 1 0.2 - 1.0 μM (final conc.)
Primer 2 0.2 - 1.0 μM (final conc.)
Sterile purified water up to 50 μl
＊ Please mix gently to be uniform and then use.
PCR conditions
This enzyme can be used in general PCR conditions, since the monoclonal antibody is denatured in the initial DNA-denaturation step. No need for a special step to denature the antibody to Taq DNA polymerase.
Note : Denaturation condition varies depending on the thermal cycler and tubes used for PCR. The recommendtion is for 5 - 10 sec at 98℃ or 20 - 30 sec at 94℃.
Competent Cell Information
DH5-alpha
Features:
A strain commonly used for plasmid cloning. The product of the φ80lacZ△M15 gene is alpha complementary with the amino terminal of the beta-galactosylase encoded by the pUC vector, which can be used for blue-white spot screening. The mutation of recA1 and endA1 was beneficial to the stability of clone DNA and the extraction of high purity plasmid DNA.
Genotype:
F- φ80lacZ△M15△ (lacZYA-argF) U169
endA1 recA1hsdR17(rk-,mk+)
supE44λ- thi-1 gyrA96 relA1 phoA
Competent Cell Information
BL21
Features:
The strain used T7 RNA polymerase as the protein expression host of efficient exogenous gene expression system. Expression of the T7 phage RNA polymerase gene is controlled by the lambda phage DE3 region of the lacUV5 promoter, which is incorporated into the BL21 chromosome. The strain is suitable for the expression of non-toxic proteins.
Genotype:
F- ompT hsdSB(rB- mB-) gal dcm（DE3）
Activity of biliverdin reductase (in vivo)
Materials
1)LB liquid medium
2)1 mM Biliverdin solution (in 100% methanol)
3)100 mM IPTG
Activity of biliverdin reductase (in vivo)
Procedures
1)Culture the E. coli cells in 5ml LB liquid medium (37℃, 220 rpm) until OD600 of the culture reaches 0.6 ~ 0.8.
2)Add 25 μl IPTG (100 mM) to the cell culture.
3)Add 5 μl biliverdin (1 mM) to the cell culture. For control group, add biliverdin after step (5).
4)Culture at 22℃, 220 rpm for 3 h.
5)Centrifuge at 10,000 rpm for 1 min.
6)Turn on the microplate reader and computer. Run the software to set the protocol and plate layout.
7)Add 100 μl of supernatant to each well on the microplate.
8)Measure the absorbance of cell culture at 600 nm as the estimation of cell amounts.
9)Measure the absorbance of cell culture at 450 nm as the amount of the product, bilirubin.
