Recovery of target DNA fragments
(1) In order to obtain the target PCR products, the target DNA fragments were separated by agarose gel electrophoresis. Generally, 1.0%agarose was used. If the DNA molecule was too small, the amount of agarose could be increased or decreased appropriately.
(2) Agarose gel electrophoresis has the function of a molecular sieve. The DNA molecule is negatively charged and moves to the positive electrode by the electric field.
(3) The size of the target band is judged by DNA Marker. The correct target band is cut with a scalpel and weighed in a 1.5 ml EP tube.
(4) The gel recovery step refers to the manual of TIANgel Purification Kit.
Escherichia coli chemistry transformation
Trans-T1 or DH5α large intestinal competent cells can be used for 2-3 transformations per tube of 100 μL stored in a refrigerator at - 80 ℃, in order to avoid the reduction of transformation efficiency during the shelf life of trans-T1 or DH5α large intestinal competent cells. The specific transformation method is as following:
(1) About 5 minutes before the end of the connection system, one sterile EP tube was pre-cooled on ice, the competent cells were put on ice, the intestinal cells were melted, and the pre-cooled yellow lance heads were used. Each EP tube was separated into another pre-cooled EP tube.
(2) Take 5-10 μL conjugated products and add them into each tube of competent cells. Attention should be paid to gently blowing two time to avoid blowing or shaking one more time, so as to avoid reducing the transformation efficiency. After adding the connecting product, put it on ice for 30 minutes, and adjust the water bath to 42 ℃ at the same time.
(3) The heat shock(42℃) time depends on the competent cells. Trans-T1 and DH5α heat shock for 45 seconds and 75 seconds respectively. After heat shock time arrives, EP tubes are quickly transferred to ice to cool the cells for 2 minutes without shaking the centrifugal tubes.
(4) Resuscitation, adding 1 mL non-resistant LB medium to each EP tube, fixing the EP tube on the float, and incubating in shaking bed at 37 ℃, 180 rpm for 60 min.
(5) 4000 rpm was centrifuged for 5 min, then part of the supernatant was removed and 100 μL supernatant was reserved for suspension. The suspension was evenly coated on the corresponding resistant LB solid plate with glass beads and cultured overnight in an incubator at 37 ℃.
Yeast transformation
In this experiment, a fragment or multiple fragments or plasmids with homologous sequences were transferred into S. cerevisiae cells by lithium acetate conversion method. The specific operational methods were as following:
(1) Inoculate a colony from a plate to 5 ml YPD liquid culture. Incubated at 30 ℃ and 200 r/min overnight.
(2) The next day, the overnight culture medium was inoculated into 5 ml YPD (usually 500 μL plus 5 ml). When incubated at 30 ℃ and 200 r/min, which required about 4-6 hrs.
(3) Take 1 ml solution, 3800 rpm centrifugation for 2 minutes, collecting cells.
(4) Resuspend and wash the cells with 1 ml sterile water, 3800 rpm centrifugation for 2 minutes and collect the cells.
(5) Resuspend cell with 1 ml 0.1M LiOAc. Put it on ice, stand by.
(6) Boil ssDNA: Placed 10mg/ml ssDNA in the PCR tube and boiled at 99.9℃ for 20~30 minutes. Put it on ice, stand by.
(7) Preparing for the transformation system:
Table 1. transformation system
(8) Added 3µL per plasmid and about 20µL ddH2O to the thallus. Added the competent cells into the transformation system and oscillated them gently to make them uniform.
(9) Cultured 30min in an incubator at 30 ℃.
(10) Heat shock at 42℃ for 18 minutes.
(11) 3800 rpm centrifugation for 2min to collect cells.
(12) Add 400μL 5mM CaCl2. Let it stand for 5 minutes.
(13) Add 700 µL YPD, culture 5h at 30 ℃, 200 r/min.
(14) 3800 rpm centrifuges for 2 min, suck out the supernatant. Add 200µL ddH2O, take 100µL coated plate..
Plasmid extraction in Escherichia coli
(1) Plasmid Extraction Kit of Escherichia coli in this paper was used to extract plasmid, the Plasmid Extraction Kit of E.coli was referred to TIANprep Mini Plasmid Kit.
(2) After the plasmid was extracted, enzyme digestion was carried out with the corresponding enzyme, and then agarose gel electrophoresis was used to see whether the size of the strip was correct.
PCR of DNA fragments
(1) Preparing for the reaction system shown in Table 2(a). The plasmid template content in the system should be within the range of 10 to 30 ng.
(2) The PCR procedure was shown in Table 3.
Table 2. Colony PCR reaction system
Table 3. The program of PCR
PCR validation of Saccharomyces cerevisiae colonies
Single colonies on the transformation plate were selected for streaking and purity (generally, 12 single colonies were selected for each transformation plate). Single colonies were selected in a 30℃ incubators for 3 days. Because yeast is an eukaryotic cell with a cell wall, colony PCR cannot be performed directly like E.coli. Yeast cell wall needs to be destroyed as a template, and the specific steps are as follows:
(1) Configure 20 mM NaOH solutions and take 30 μL into each PCR pool.
(2) Use toothpicks to pick a small number of cells into the PCR pools, the solution will become slightly cloudy, too many cells will not break the cell walls thoroughly.
(3) Boil at 99℃ for 5 min and 4℃ for 1 min on the PCR instrument for 3 cycles, and put in the refrigerator at 4℃ after the end as the template bacteria liquid.
(4) Colony PCR was performed with the prepared template bacteria liquid, and the PCR system was shown in Table 2(b). The PCR procedure was shown in Table 3.
E. coli Colony PCR Experiment Procedure
(1) Cultivate individual colonies at 37°C in an incubator for 3 days. Typically, select 12 individual colonies from each transformation plate.
(2) Add 10μL of ddH2O into a PCR tube.
(3) Use a toothpick to pick a small amount of cells and place them into the PCR tube. The solution will become slightly cloudy.
(4) Pipette 1μL from the solution to use as the PCR template.
(5) Perform colony PCR using the prepared bacterial liquid template. The PCR reaction system and procedure are the same as yeast colony PCR.
Restriction Enzyme Digestion of DNA
(1) Different restriction endonucleases identify different restriction sites and cut the target fragment, so that the target fragment has sticky ends. They provide carriers or fragments for subsequent experiments to verify whether the correct transformer is correct and select the correct size of the band for testing.
(2) Double enzymes have high efficiency of ligation, while single enzymatic digestion can easily lead to carrier self-ligation, which reduces the efficiency of ligation.
(3) The restrictive endonucleases of NEB and Thermo FastDigest was mainly used in this experiment. The buffer and temperature of the reaction should be used according to the instructions of each enzyme, otherwise the efficiency of enzymatic digestion would be reduced.
(4) Generally speaking, NEB Enzymes can be digested overnight without asterisk activity; Thermo FastDigest belongs to fast digestion enzymes, generally 2~2.5h can be used, too long digestion time will have Asterisk activity.
(5) The enzyme digestion reaction system is shown in Table 4.
Table 4. Restriction enzyme digestion system
Method for Making E. coli Test Paper
(1) Yeast cultures grown in LB liquid medium with an optical density (OD600nm) ranging from 1.2 to 4.2 were centrifuged at 3700g for 10 minutes to concentrate the cells. The supernatant was removed, and E. coli cells were suspended in 2% low-viscosity sodium alginate containing 5% sucrose. Each 10μL solution contained E. coli cells with a concentration equivalent to 2.0 OD600nm units.
(2) The E. coli-sodium alginate solution (10μL) was spotted in the center of the test paper. Subsequently, the test strips were immersed in a 4% CaCl2 solution for 15 minutes for cross-linking. Before conducting the test, the cross-linked test strips were rinsed with deionized water and air-dried for at least 2 hours at room temperature.
Test Paper Effectiveness Testing Procedure
(1) Dilute IPTG to concentrations of 1.4mg/L, 2.8mg/L, 14mg/L, and 70mg/L. Dilute tetracycline to concentrations of 2mg/L, 4mg/L, 20mg/L, and 100mg/L.
(2) Apply 100μL of different concentrations of the fluorescent inducer solutions onto the test strips, ensuring that the detection area of the test strip is completely soaked in the inducer solution. Seal the test strips in a petri dish, maintain humidity using a sponge block saturated with 1 cubic centimeter of water, and store at 37°C for 24 hours.
(3) After the incubation, observe the fluorescence intensity in the detection area of the test strips, and record the fluorescence reaction results for each concentration. This experimental design allows you to test the fluorescence response of the test strips at different concentrations, helping you understand the sensitivity and stability of the test strips for different inducer concentrations.
Goldengate-Assembly Protocol
(1) Construction of sgRNA Vector:
The sgRNA vector was constructed using BsaI restriction endonuclease. Initially, DNA concentrations of the insert fragment and the vector plasmid were measured using a spectrophotometer and diluted to 25 fmol each. The pH of HEPES buffer was adjusted to 7.8 using sodium hydroxide solution. The primer system used is detailed in Table 5.
(2) Primer Annealing:
The primer system was subjected to annealing by placing it in a PCR thermocycler. The program was set to 95°C for 5 minutes, followed by a gradual decrease to 4°C at a rate of 0.1°C/s, and then maintained at 4°C for 15 minutes. Subsequently, 120 μL of ddH2O was added to dilute the mixture to the desired concentration.
(3) Reaction Setup:
The reaction mixture was prepared by adding insert fragments, vector, enzymes, and buffer solution according to Table 6.
Following the preparation of the reaction mixture, the Goldengate reaction was carried out according to Table 7. Specific conditions and steps were determined based on the laboratory's operational manual or experimental protocol.
Table 5
Table 6
Table 7
Seamless Cloning in Escherichia coli
(1) Seamless Cloning in this paper was used to seamless cloning. The seamless cloning assembly kit of E.coli was referred to pEASY®-Basic Seamless Cloning and Assembly Kit.
(2) Prepare the reaction system as Table 8. The optimal molar ratio between the vector and each insert fragment is 1:2.
(3) The linear vector fragment and Basic Assembly Mix were mixed and reacted at 50℃ for 20min.
Table 8
