Note
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-culture the competence cell
-test the experiment kits -
-Molecular cloning for Bba_J06602, BBa_B0012, Bba_K592024, Bba_I13453, Bba_B0010
-Tried to reverse pBAD(BBa_I13453)
-preparation of the target bio-brick -
-Ligation: flpe-BFP, loxP-pBAD, B0010-loxP, eGFP-B0015, FRT-pBAD -Preservation of the plasmid pAUR135 in E.coli; Transformation of the commercial plasmid pAUR135(from TAKARA, Item No.D3604); select monoclonal colony and extract plasmid(pAUR135) from the culture. -Prepared Yeast Extract Peptone Dextrose(YPD) Medium
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Method
Preparation of E. coli competent cell
Wild type E. coli DH5α, BL21 (DE3), or Top10 is inoculate on a LB plate and incubated in 37℃ overnight. Single colony is inoculated into 3 mL LB broth for overnight growth. This culture is 1:100 inoculated into a second 50 mL LB and let growth for 2-5 h until OD600 reach around 0.5. The culture is then ice-bathed, centrifuged and resuspended by 50 mL 10% glycerol with 0.1 M CaCl2 for twice, and all the way 4℃. Finally, the bacterial cells are resuspended into 7 mL solution of 10% glycerol with 0.1 M CaCl2, transferred into each EP tube for 120 μL, and stored at -80℃.
PCR
We use 3 kinds of DNA polymerase to deal with different situation, that is, Takara PrimeSTAR max (Cat. R045A), Vazyme Phanta max (Cat. P515), or Genstar Taq (Cat. A112). The protocol refers to the instruction of corresponding enzyme. The product is purified through electrophoresis in 1.0-2.0% agarose gel and OMEGA Gel Extract Kit (Cat. D2500-02).
Preparation of Plasmid DNA
At least 4 mL culture of E. coli DH5α is gathered after 16-18 hours of growth in LB with proper antibiotics. The plasmid is purified through Genstar StarPrep Plasmid Miniprep Kit StarPrep (Cat. D201-01) or OMEGA Plasmid Mini Kid I (Cat. D6943-02).
Digestion
20 μL (about 1.5 μg plasmid or 1 μg PCR purified product) substrates are added into totally 50 reaction solution, with at least 1 μL each restriction enzyme (NEB). After incubation for at 37℃ for least 1.5h, the product is purified through electrophoresis in 1.0-2.0% agarose gel and OMEGA Gel Extract Kit (Cat. D2500-02).
Gel Purification
We use HiPure Gel Pure DNA Micro Kit (Cat. D2110-01) offered by Magen to purify DNA. It is suitable for a variety of routine applications including sequencing and ligation.
Ligation
The ligation is directed by 3A assembly. Add 25ng Fast DNA-Backbone, appropriate DNA-fragment (The amount of substance of DNA-fragment shall be 3-10 times as much as DNA-Backbone), and 1μL T4 Buffer 10 X, 1μL T4 ligase into a PCR tube. Then add MINI-Q water to ensure that the volume of the mix is 10μL. Incubate the tube at 4℃ overnight.
Yeast
- Grow yeast cells overnight in YPD
- Harvest cells by centrifugation
- Resuspend cell pellet in 5 mL TE + 0.1 M lithium acetate and centrifuge.
- Resuspend cell pellet in 2 mL TE + 0.1 M lithium acetate. Incubate at 30℃ for 1 h and rapidly chill on ice
- In a tube, add Plasmid DNA for transformation, 0.2 mL cells treated with LiOAc from above and 1 mL 40% PEG 4000, 1XTE pH 7.5, 0.1 M Lithium acetate.
- Mix by vortexing and incubate at 30 degrees for 30 min.
- Heat shock cells for 15 min. at 42 degrees.
- Shortly centrifuge and remove supernatant
- Resuspend cell pellet in 1 mL TE
- Shortly centrifuge and remove supernatant
- Resuspend cell pellet in TE by vortexing.
- Plate to selective plates.
E.coli
Transformation must be conducted at once after ligation is over. 20 μL ligation product or 1-2 μL plasmid is added to about 120 μL E. coli competent cell, and incubated on ice for at least 20 min. Transfer the solution into 42℃ and keep for 80s, and return to ice and keep for 3 min. Afterward, add 700 μL LB into each tube and shake for 220 rpm at 37℃, for 30-60 min. Finally, centrifuge and remove 600 μL LB, resuspend the bacterial and spread them on LB plates with proper antibiotics.
Strain preparation
Inoculate a strain into 3 mL LB with 2 proper antibiotics (for us, Km and Cm) and cultivate them overnight. This culture is 1:100 inoculated into a second 8 mL LB (in Φ18mm tube), and pre-shaking for 2 h 30 min.
Induction
For T7-lacO promoter in BL21 (DE3), add IPTG at final concentration of 125nM; for PBAD promoter in Top10, add arabinose at final concentration of 1mM. The measurement starts from this point.
Sampling & measurement
At each sampling time point, transfer 200 μL into a well of 96-well plate, 2 parallel tubes for each strain. When adding samples finished, immediately return the tube to the shaker. For each well we read OD 700, OD 600, eGFP fluorescence 480/515, and mCherry fluorescence 580/615 using a multi-function plate reader. Usually we pick sample and measure the data at 0 h, 1 h, 2 h, 3 h, 4.5 h, 6 h, 8 h, 10 h, 12 h, 15 h, 19 h, and 24 h for each strain.
Data process
The background value of LB is subtracted from each well, then average value of OD 700, OD 600 (usually useless for us), eGFP RFU, and mCherry RFU for each sample is calculated. Finally, we use RFU per OD 700 as data to describe the dynamics of invertase in certain number of bacteria. This data is further analyzed by our modeling work (这里超链接指向建模method).
Measuring fluorescence with microplate reader
Colony PCR for positive check
Prepare Taq PCR solution without any template. Add 15 μL such solution to each PCR tube and pick a tiny spot of a colony and dip it into the PCR tube and then start PCR. For this method, the PCR pre-denature step (more than 94℃) should be more than 5 min.
- Grow yeast culture in YPD medium and harvest by centrifugation at 4000x g for 10 min at room temperature.
- Discard medium and resuspend cells in 480 μL Buffer SE, 10 μL 2-mercaptoethanol and 20 μL lyticase solution. Incubate at 30℃ for at least 30 min.
- Pellet spheroblast by centrifuging 5 min at 4000x g at room temperature.
- Add 200 μL Buffer YL and 50 mg glass beads to the sample. Vortex at max speed for 3-5 min. Let it stand to allow the beads to settle. Transfer supernatant to a new 1.5 mL centrifuge tube.
- Add 25 μL proteinase K solution and vortex to mix well. Incubate at 65℃ in a shaking water bath for 30 min.
- Add 5 μL RNase A to the sample and invert tube several times to mix. Incubate at room temperature for 10 min.
- Add 220 μL Buffer YDL and 220 μL absolute ethanol to the sample and mix thoroughly by vortexing at maxi speed for 20s.
- Assemble a HiBind DNA Mini Column in a 2 mL collection, Transfer the entire sample from Step 8 into the column, including any precipitate that may have formed. Centrifuge at 10000x g for 1 min to bind DNA. Discard the collection tube and filtrate.
- Place the column into a second 2 mL tube and wash by adding 500 μL buffer HB. Centrifuge at 10000x g for 1 min. Discard flow-through and reuse the collection tube.
- Place the column into the same collection tube and wash by adding 700 μL DNA Wash Buffer diluted with ethanol. Centrifuge at 10000x g for 1 min. Discard flow-through and reuse the collection tube.
- Wash the column with a second 700 μL DNA Wash Buffer and centrifuge as above.
- Using the same 2 mL collection tube, centrifuge HiBind DNA Mini Column at maxi speed for 2 min to dry the column.
- Place the column into nuclease-free 1.5 mL microfuge tube and add 50-100 μL of preheated Elution Buffer to HiBind DNA Mini Column matrix.
- To elute DNA from the column, centrifuge at 10000x g for 1 min.
Program:
Add 1μL Dpn I and cultured in 37℃ for 1h.Transform 10 μL product into 50 μL Top10 (competent cell) and cultivate in 37℃ overnight. Select monoclonal colony for plasmid extraction.
- Prepare 10x S1 nuclease buffer:
- 300 mM Sodium acetate, pH 4.6
- 2800 mM NaCl
- 10 mM ZnSO4
- Selective degradation of single-stranded DNA:
- incubate at 23°C, 15min
- Add EDTA
- Ligation with T4 DNA polymerase:
- Incubate at 22℃ for 1 h
- Incubate at 65℃ for 10 min to inactivate the enzyme
- Transformation and plasmid extraction.
- Double enzyme digestion (HF，NEB)
- Incubate at 37℃ for 2 h
- Purify digestion product with Gel extraction kit (Tiangen)
- Anneal of MCS oligonucleotide (MCS anneal product )
- Ligation
- Incubate at 22℃ for 1h
- Transformation and plasmid extraction.
- Insert DNA fragment into pAUR135 vector
- Transform yeast cells by acetate lithium method
- Culture in YPD medium for more than 6h and spread onto YPD selective medium plate containing AbA
- Select resistance marked yeast cells.
A strain of overnight culture can be stored by adding glycerol at a final concentration of 15-20% and keeping them at -80℃.
Parts
Additionally, we prepared a website plug-in to for potential users to design their own timer with specific length of counting time, a project in cooperation with SYSU-software. According to the data gathered in this research and other promoter intensity given by iGEM official page, we can anticipate the overall timing length of any given combination of various invertase module. Vice versus, if a user could provide his/her target time, we can automatically generate one or more optimized design of Micro-time to precisely match the demand.
