Team:HZAU-China/Protocol
index
. Molecular Cloning
o DNA Synthesis
Primer synthesis service is provided by Sangon Biotech (Shanghai) Co., Ltd.
Gene synthesis service is provided by Genscript.
o PCR
Regular PCR, OE-PCR were carried out with PrimeSTAR® Max DNA Polymerase from TaKaRa and the protocol it provided online. PCR products with similar length to its template were digested with QuickCut Dpn I from TaKaRa and the protocol it provided.
Colony PCR were carried out with 2×Taq PCR MasterMix from Aidlab Biotechnologies Co.,Ltd and the protocol they provided. The solution system we used was 10μL. The initialization and denaturation temperatures were set at 95℃.
o Homologous Recombination
Two-fragment recombination were carried out with ClonExpression® II One Step Cloning Kit from Vazyme biotech co., ltd and the protocol it provided.
Three-fragment recombination were carried out with ClonExpression® MultiS One Step Cloning Kit from Vazyme biotech co., ltd and the protocol it provided.
o Gibson Assembly
Gibson assembly were carried out according to the protocol below.
1) Prepare 6 ml of 5X ISO Buffer in a 15 mL falcon tube as follow:
| 3 mL | 1 M Tris-HCl pH 7.5 | |
|---|---|---|
| + | 150μL | 2 M MgCl2 |
| + | 240μL | 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP) |
| + | 300μL | 1 M DTT |
| + | 1.5 g | PEG-8000 |
| + | 300μL | 100 mM NAD |
| + | ddH2O to | |
| 6 mL |
Store at -20℃ in 320μL aliquots.
2) Prepare 1.2 mL of Gibson assembly master mix as follow:
| 320μL | 5X ISO Buffer | |
|---|---|---|
| + | 0.64μL | 10 U/μL T5 exonuclease |
| + | 20μL | 2 U/μL Phusion polymerase |
| + | 160μL | 40 U/μL Taq ligase |
| + | ddH2O to | |
| 1.2mL |
Store at -20℃ in 15μL aliquots.
3) Thaw a 15μL aliquot of the Gibson assembly master mix and keep on ice until use.
4) Measure the DNA concentration (ng/μL) of each assembly piece.
5) Add 0.02×length (ng) of each linearized fragment to the thawed 15μL master mix in a 20μL total volume assembly reaction mixture as follows:
| assembly fragments | ||
|---|---|---|
| + | 15μL | Gibson assembly master mix |
| + | ddH2O to | |
| 20 μL |
6) Incubate the assembly reaction at 50℃ for 60 minutes, and then place on ice.
T5 Exonuclease,Taq DNA Ligase and Phusion® High-Fidelity DNA Polymerase are obtained from NEW ENGLAND BioLabs® Inc.
. Competent Cell
Commercial competent cells were obtained from Shanghai Weidi Biotechnology Co., Ltd.
Self-made competent cell was made according to this paper:
Im H, Sambrook J, Russell D W. The inoue method for preparation and transformation of competent E.coli: "Ultra Competent" cells[J]. Bio-protocol Bio101, 2011.
. Transformation
The protocol we used to transform plasmids into competent cells are shown below:
1. Melt the competent cell on ice for 5 minutes.
2. Add proper amount of plasmid into the competent cell and place it on ice for 25 minutes.
3. Heat shock for 45-90 seconds. Place the tube on ice immediately for 2-3 minutes.
4. Add antibiotic free LB into the competent cell. Incubate for 1 hour, 37℃, 200rpm.
5. Coat the competent cell on a LB agar plate with proper antibiotic.
6. Incubate at 37℃ overnights.
. Smell-sensing Module Verification
1. Methods of molecular cloning and transformation are described above.
2. Transform this part into E. coli DH5α and pick three monoclonal from the same plate as biological repeats. 10ml LB medium containing 170μg/mL chloramphenicol was used and the seed culture were grown at 37℃ while shaking at 200 rpm overnight.
3. Dilute the seed culture to 10ml with LB at a ratio of 1:100. Culture the cells as above described until OD600=0.3, then different concentrations of benzyl alcohol were added (0 mM, 0.01 mM, 0.1 mM, 1 mM, 2.4 mM, 3.8 mM, 5.3 mM) and three parallel repeats were set for each concentration.
4. Shake the bottle to make the benzyl alcohol fully dissolved, then add the sample to a 96-well plate and cultivate in Synergy H1 hybrid multi-mode reader with 37℃, 180cpm. The OD600 value and the fluorescence value were determined every 30 minutes for 8 hours.
. Memory Module Verification
o taRNA-crRNA interaction
1. Methods of molecular cloning and transformation are described above.
2. Transform the plasmids with taRNA and crRNA into DH5α. Then spread them onto a LB plates with 60μg/mL ampicillin, respectively.
3. Pick an isolated colony with a sterile tip from the LB plates and add each tip into 5ml LB medium with 60μg/mL ampicillin. Incubate for overnight at 37℃ in a shaker with 200 rpm.
4. Dilute the bacterial fluids to OD≈0.3 to form a 20 mL total volume, then incubate for another 4 hours.
5. Divide each 20mL medium to two 10mL medium. Add 100μL 100mM IPTG into one of the two 10mL medium. The other one remains as negative control.
6. Induce for 4 hours. Then measure the two sample's fluorescence intensity (excitation: 485nm, emission: 528nm) and OD600 in the plate reader (Synergy H1 hybrid multi-mode reader) using a 24-well transparent plate. Each well is filled with 1mL sample.
7. Data taken from the plate reader are analyzed using Excel.
8. All experiments above are carried out in 3 biological replications.
o Positive Feedback
1. Methods of molecular cloning and plasmid construction are described above.
2. Transform these plasmids into E. coli DH5α and pick three monoclonals from the same plate as parallel repeat, 5mL LB medium containing 20μg/mL ampicillin and 10nM AHL is used and the seed culture media are grown at 37℃ while shaking at 200rpm overnight.
3. Dilute the seed culture medium to OD600 = 0.1 (diluted about 10 times) and then add them into 96-well plate, each sample is added to one hole. Cultivate in Synergy H1 hybrid multi-mode reader with 37℃, 180cpm. The OD600 value and fluorescence value are detected each 20 minutes in 8 hours.
4. The blank control is the same volume of LB medium containing 20μg/mL ampicillin.
o The Switch
1. Methods of molecular cloning and plasmid construction are described above.
2. Transform this plasmid into E. coli DH5α and pick three monoclonals from the same plate as parallel repeat, 5mL LB medium containing 20μg/mL ampicillin and 10nM acylated homoserine lactones (AHL) are used and the seed culture media are grown at 37℃ while shaking at 200rpm overnight.
3. Dilute the seed culture medium to OD600= 0.1 and then add into 96-well plate, each sample is added to two holes, one adds 1mM IPTG and the other adds the same volume of water. Cultivate in Synergy H1 hybrid multi-mode reader with 37℃, 180cpm. The OD600 value and fluorescence value are detected each 20 minutes in 8 hours.
4. The blank control is the same volume of LB medium containing 20μg/mL ampicillin.
. Reproduction Module Verification
1. The plasmid was constructed by Gibson assembly. Methods of transformation are described above.
2. LB was supplemented with 20g/L D-glucose, 0.2g/L L-tyrosine, 0.1g/L L-tryptophan and 3g/L L-aspartic.[1]
3. E. coli BCAE (W3110 ΔtyrB::FRT, ΔaspC::FRT, tyrA16::Tn10, ΔtrpE::FRT) within pSUFAAQ (pSU2718 derivative, aroFfbr,pheAfbr, lacIq, hmaSAo) provided by Zhoutong Sun and Sheng Yang from CAS Center for Excellence in Molecular Plant Sciences/Institute of Plant Physiology and Ecology was transformed with vcm17-mdlC-mdlB. Single colony was selected from the transformant pool and seed cultures were grown in 10 mL LB broth with 20μg/mL ampicillin at 37℃ while shaking at 200rpm overnight. Each seed culture was used to inoculate 10 mL LB and appropriate antibiotics in a 20mL bacteria flask.[1]
4. Upon reaching an optical density at 600nm (OD600) of 0.7, cultures were induced by addition of 1mM IPTG.
5. Samples were prepared by centrifuging 1 mL of culture at 10,000g for 3 min to pellet and remove cells. Supernatants were transferred to an HPLC vial. HPLC analysis was performed using a DAD3100 HPLC instrument within a Supersil ODS2 5μm column. Samples (20μL) were injected into a mobile phase with a constant total flow rate of 1mL/min. The mobile phase consisted of 55% "solvent A" consisting of nanopure water, and 45% "solvent B" consisting of HPLC-grade acetonitrile.[2]
6. Three biological repeats were performed to provide estimates of standard error.
. Erasure Module Verification
1. Methods of molecular cloning and plasmid construction are described above.
2. Transform this plasmid into E. coli DH5α and pick three monoclonals from the same plate as parallel repeat, 5mL LB medium containing 20μg/mL ampicillin and 10nM acylated homoserine lactones (AHL) is used and the seed culture media are grown at 37℃ while shaking at 200rpm overnight.
3. Dilute the seed culture medium to OD600 = 0.1 and then add them into the 96-well plate, each sample is added to two wells, one adds 100ng/mL anhydrotetracycline (ATc) and the other adds the same volume of ethyl alcohol. Cultivate in Synergy H1 hybrid multi-mode reader with 37℃, 180cpm. The OD600 value and fluorescence value are detected each 20 minutes in 8 hours.
4. The blank control is the same volume of LB medium containing 20μg/mL ampicillin.
. Parts Improvement
1. Methods of molecular cloning and transformation are described above.
2. Transform four different plasmids with low copy ampicillin resistance into E. coli DH5α, which are named 1, 2, 3, 4 respectively. One base pair is mutated in plasmid 1 and 2. Two base pairs are mutated in plasmid 3. Plasmid 4 is the plasmid with the original part BBa_R0062. Then spread them onto four LB plates with 20μg/mL ampicillin, respectively.
3. Pick four isolated colonies with a sterile tip from each of the four LB plates and add each tip into 5ml LB medium with 20μg/mL ampicillin. Incubate for 6-8 h at 37℃ in a shaker with 200rpm.
4. Add each kind of bacterial fluid into different lines of a sterile, black-coated 96-well plate. Add shaken bacterial solution and fresh medium with ampicillin to a total of 100μL, and make sure the OD600 of each well is close to 0.1, measured by the plate reader.
5. Add 1μL AHL diluted in DMSO into each well. The final concentration range of AHL is from 10-8 to 1 mmol/L. Add 1μL DMSO without AHL served as positive controls. Blank controls are fresh LB medium. Place the 96-well plate into the automatic microplate reader (Synergy H1 hybrid multi-mode reader). Incubate at 37℃ overnight and measure the fluorescence value（excitation: 485nm, emission: 528nm）and OD600 of each well every 30 minutes.
6. Data taken from the plate reader are exported to Excel and imported to Prism for analysis.
7. All experiments above are carried out in 3 biological replications.
. Part Characterization
1. Methods of molecular cloning and transformation are described above.
2. Transform three different plasmids with high copy chloramphenicol resistance into E. coli DH5α, which contain J23102, J23105, J23109 respectively. All plasmids contain BBa_C0062 which is the part we want to characterize. Then spread them onto three LB plates with 170μg/mL chloramphenicol, respectively.
3. Pick three isolated colonies with a sterile tip from each of the three LB plates and add each tip into 5ml LB medium with 170μg/ml chloramphenicol. Incubate overnight at 37℃ in a shaker with 200rpm. Take 100μL bacterial of each flask into 5mL fresh new LB and incubate for another 3-4 hours.
4. Add each kind of bacterial fluid into different lines of a sterile, black-coated 96-well plate. Add shaken bacterial solution and fresh medium with 170μg/ml chloramphenicol to a total of 100μL, and make sure the OD600 of each well is 0.02-0.03, measured by the plate reader.
5. Add 1μL AHL diluted in DMSO into each well. The final concentration range of AHL is from 10-8 to 0.1mmol/L. Add 1μL DMSO without AHL served as positive controls. Blank controls are fresh LB medium with DH5α. Place the 96-well plate into the automatic microplate reader (Synergy H1 hybrid multi-mode reader). Incubate at 37℃ overnight and measure the fluorescence value（excitation: 485nm, emission: 528nm）and OD600 of each well every 30 minutes.
6. Data taken from the plate reader are exported to Excel and imported to Origin for analysis.
7. All experiments above are carried out in 3 biological replications.
Reference
[1] Sun Z, Ning Y, Liu L, et al. Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S-or R-mandelic acid[J]. Microbial Cell Factories, 2011, 10(1): 71.
[2] Pugh S, McKenna R, Halloum I, et al. Engineering Escherichia coli for renewable benzyl alcohol production[J]. Metabolic Engineering Communications, 2015, 2: 39-45.
