Team:Tianjin/Notebook
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Construct a Ura-tagged GFP frequently expressed fragment and transfer it to SY14
May 10 Yeast transformation, transfer Cas9 into SY14 cells to obtain SY14-Cas9 cells Obtain 6 GFP fragments promoter P, left homology arm L, right homology arm R, terminator T, GFP main body G fragment, and Ura fragment from PRS416; May 13 Overlap connects the following fragments of GFP: Group A: P and T segments; Group B: U and R segments; Group C: L and P fragments; Group D: G and T segments; Group E: L-P-G-T fragment; And full-length fragments; PCR verifies whether the above fragments are connected. Result: P-G-T; U-R; G-T; L-P-G-T connected correctly. Culture SY14-Cas9 cells. Yeast transformation: Control group: transfer Leu plasmid into SY14-Cas cells; Experimental group: Delta6 plasmid was transferred into SY14-Cas cells. May 14 to May 29 Overlap connect B and E fragments and verify by PCR; Overlap connect L-G-U-R fragments and verify; Correct fragments are glued and stored; Verify whether Leu plasmid is transferred to SY14-Cas cells by PCR and agarose gel electrophoresis; It was verified by PCR and agarose gel electrophoresis that the Delta6 plasmid was transferred into SY14-Cas cells. May 29 Get full-length GFP. Transform the ligated full-length GFP plasmid into yeast SY14-Cas cells. May 30 to June 4 Overlap repeats the connection work of each fragment. Connect the repeatedly connected fragments and transfer them into yeast SY14-Cas cells. Verify whether the GFP plasmid has successfully entered the cell by PCR and agarose gel electrophoresis. June 4 It proved that the plasmid transferred into the cell may not exist stably, and the induction operation was not good, so it was decided to abandon the construction of the experimental plasmid.
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Construct the GFP2.0 frequently expressed fragment of ADH1 promoter and transfer it into SY14-Cas yeast
June 5 Replace the promoter in GFP1.0 to construct plasmid GFP2.0 Connection (the following are all 2.0 fragments) Group A: P and T segments; Group B: U and R segments; Group C: L and P fragments; Group D: G and T segments; Group E: L-P-G-T fragment; Overlap group E and group B and verify. June 6 Get the full-length GFP2.0 fragment after verification, the result is correct, and the gel will be collected Transform the obtained GFP2.0 plasmid into yeast SY-14 Overlap repeatedly connects each group of fragments. June 7 to June 29 Overlap repeatedly connects each group of fragments Overlap repeatedly connects group E and group B, group D and group B to obtain the full-length plasmid GFP2.0 Transform each full-length fragment GFP2.0 into SY14-Cas yeast and perform PCR verification July 5 It was verified that the full-length GFP2.0 plasmid transferred into yeast SY14-Cas on June 29. The L, P, R, T, and G fragments were successfully ligated to the plasmid, but the Ura tag was not ligated, and subsequent ligation of the fragments and transformation were required. July 8 Overlap connect the full length GFP2.0 fragment, F-U-R fragment, U-R fragment and verify. T-U-R and U-R are successfully connected. July 8 to July 14 Link the obtained T-U-R fragment and U-R fragment with other fragments July 15 Verify GFP-Ura, the experiment is correct, it proves that the Ura tag is successfully inserted into the plasmid, and the complete plasmid GFP2.0 is obtained July 17 to July 19 Transfer the obtained GFP2.0 into yeast cell SY14-Cas Transform the obtained GFP-Ura into yeast cells SY14-Cas The verification proved that GFP2.0 was successfully transferred into yeast cell SY14-Cas, and GFP-Ura was successfully transferred into yeast cell SY14-Cas. Store the above two kinds of cells at low temperature. This step is all completed.
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Construct a fragment of fGFP that can be induced to rapidly degrade and transfer it to 4742 cells
August 23 Obtain Escherichia coli containing fGFP fragments from the laboratory and store them in solid medium. Extract the fGFP plasmid. The fGFP fragment was transferred into yeast cells containing empty plasmids. The fGFP fragment was transformed into yeast cells containing Delta-g. The fGFP fragment was transformed into yeast cells containing 7flip-Cre. August 24 to August 30 The presence of fGFP fragments in yeast cells was verified by PCR and agarose gel electrophoresis. And ligate the ligated fGFP-Cre with the empty plasmid. Obtain FGFP-empty-Cre cells.
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Construct Delta-g plasmid and transfer it into SY14
May 28 to July 13 Construction of the cutting plasmid Delta-g: Copy the fragment from the original vector PRS42H plasmid (from the laboratory) by PCR and agarose gel electrophoresis; Copy the Cas9 fragment from the Cas plasmid by PCR and agarose gel electrophoresis; Cooperate with the company to synthesize sgRNA sequence; Copy the gal promoter from laboratory cells by PCR and agarose gel electrophoresis. July 14 The above-mentioned fragments were transferred into yeast cells SY14 and connected in yeast cells. The Delta-g plasmid is obtained. July 18 Transfer Delta-g to yeast 4741; Transfer Cas9 to yeast 4741. July 20 The Delta-g plasmid was transformed into yeast cells containing GFP2.0. July 22 to July 31May 28 to July 13 Transform the Delta-g plasmid into yeast cells SY-14 containing GFP2.0; Transform the Delta-g plasmid into yeast cells SY-14 containing GFP-Ura; Repeat the above operation and verify with PCR, transfer the obtained correct cells at regular intervals to ensure the viability of the cells. August 1 to August 9 Due to the poor viability of SY-14 cells, we chose to replace 4741 yeast cells in subsequent experiments. Transform Delta-g plasmid and GFP2.0 into yeast cell 4741; Transform Delta-g plasmid and GFP-Ura into yeast cells 4741. Verify the cutting efficiency of Delta-g plasmid. The SY-14 yeast and the 4741 yeast containing Delta-g were respectively transferred to the medium containing gal and the medium without gal as the experimental group and the control group. The gal induced the expression of Delta-g and cut the chromosome. After 23 hours of incubation, the OD of the experimental group and the control group were measured and diluted by the same multiple (104, 103, 102) to plate the culture. We observed that the cut cells all lost GFP, which can prove to a certain extent that GFP was cut down. However, in the follow-up experiments, as the culture time increased, the OD of the gal-induced experimental group gradually approached or even exceeded the non-gal-induced control group, so we speculated that the cell chromosomes were not knocked out, but the escape of the plasmid occurred, so This plasmid cannot complete the subsequent construction of chromosome-free cells. Therefore, the Delta-g project was terminated.
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Construct 7flip-Cre plasmid and transfer it into 4742
August 20 Obtain the 7flip-Cre plasmid from the laboratory. August 23 to August 30 The fGFP was transformed into yeast cells 4741 containing the 7flip-Cre plasmid. The cleavage plasmid fGFP-7flip-Cre required by chromosome-free yeast cells was constructed.
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Construct the tetR-Cas9 plasmid and transfer it into 4742
September 9 The tetR-Cas9 plasmid was obtained from the laboratory. Connect with Delta to obtain Delta-tetR-Cas9 plasmid.
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96-well plate growth experiment
October 1-7 During flow sorting, the chromosome-free cells/normal control cells are directly divided into the appropriate medium, 1- 5 per well (unknown). In theory, the growth status of the control group is normal, and the experimental group can hardly grow.
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Demolition
September 11 to October The sorted achromosome cells and normal cells were separated by a cell splitter to separate dozens of single cells and cultured. Theoretically speaking, the growth status of the control group was normal, and the experimental group was basically unable to grow.
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CFU
September 11 to October 1 Repeat the chromosome-free cell preparation experiment, and measure the OD at 48 hours, 60 hours, and 72 hours after induction, and then dilute and plate them. The dilution factor is 104, 105, 106. Count the number of colonies on the solid medium 2 to 3 days after each coating, and record the experimental data to complete the comparison.
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fGFP indicates no chromosome formation
The fGFP in the control group is normally expressed and located on the genome, while the chromosome-free cells in the experimental group do not have fGFP and cannot produce fluorescence. Due to the short half-life of fGFP, after sorting, the green fluorescence intensity is measured by taking pictures, and the green fluorescence in the control group The brightness is stable, and the experimental group gradually darkens with the extension of the culture time until the fGFP is completely degraded and there is no bright light.
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Nucleic acid staining
After elapsed cell sorting, the cells are stained with nuclear-specific binding dyes. The nucleus of the control group of chromosomal cells does not disappear and a bright light spot appears, while the cells of the non-chromosome experimental group show diffuse and weak fluorescence. By taking photos/videos, it can be verified that there is no chromosome formation.
Preliminary Preparations
(1) Prepare YPD solid medium (2) Obtain the original cells, including 4741 (4741A), 4742 (4741α), and SY14.After obtaining the YPD medium, the solid plate is streaked. (3) For bacteria to be stored in glycerol, new plates should be drawn regularly (1- 2 weeks) to maintain the vigor of the bacteria. (4) Collection of various consumables, cleaning and recycling of test tubes, glass beads, large pipette tips, conical flasks, and reagent bottles. (5) Laboratory cleaning and finishing work, necessary reagents and common mother liquor (such as: deionized water, amino acid mother liquor, sodium hydroxide, etc.) equipped with pipette tips, toothpicks, reagents, culture medium, centrifuge tube sterilization.
Construction of fGFP MAY 10 ~ AUGUST 30
The main content of this experiment is to continuously update the original to construct the GFP plasmid, so as to realize the iteration of GFP. We first use GFP with an unknown promoter to construct a plasmid and transfer it into the cell to make it often expressed. However, because the plasmid is difficult to stably exist and is ethanol-inducible, the effect is poor in subsequent induction experiments, so we abandoned this plasmid. So we replaced the promoter and used ADH1 to control its transcription to get GFP plasmid 2.0. In subsequent experiments, we found that GFP has a very stable expression intensity under the condition of normal expression, and it can still maintain a high fluorescence brightness after the cell loses the chromosome, which will affect the follow-up observation of whether the yeast cell has completed the chromosome excision. We chose to construct the fGFP plasmid. The fGFP plasmid can ensure that fGFP is normally expressed in chromosome yeast cells, while chromosome-free cells do not have fGFP and no longer produce fluorescence. Because the half-life of fGFP is short, after sorting, the brightness of the control group's gfp will be stable, while the experimental group It will get darker and darker until the fGFP is completely degraded and there is no bright light. It is more convenient for the characterization of experiments. The following will introduce the experimental process based on the three GFP plasmids.
Construction of carotene plasmid MAY 17 ~ SEPTEMBER 23
May 17 Extract the carotene plasmid and transfer it into SY14 cells. May 19 to May 27 Verify whether carotene is transferred into cells by PCR and agarose gel electrophoresis. May 31 Repeat the transfer of the carotene plasmid into SY14 cells. June 5 Verify whether the carotene plasmid is successfully transferred into SY14 cells. June 30 Repeat the transfer of the carotene plasmid into SY14 cells. July 1 Extract the carotene plasmid in Escherichia coli. July 2 to July 4 Since the transfer of the carotene plasmid into yeast was not good, in order to preserve the carotene plasmid, the method of electrotransformation of Escherichia coli was adopted. The plasmid was first transferred to Escherichia coli for storage to obtain Escherichia coli 001 and E. Method, transfer the obtained plasmid into CREATE empty yeast cells. July 8 The plasmid in E. coli 002 was extracted and transferred to yeast. July 12 Yeast did not produce carotene, and PCR did not verify the presence of a carotene plasmid in yeast. July 21 to July 22 Transform tetR into E. coli 002, and verify by PCR and agarose gel electrophoresis. No good experimental results were obtained. July 26 to July 27 Transform tetR into E. coli 002, and verify by PCR and agarose gel electrophoresis. The assembled carotene plasmid is obtained. July 29-August 1 The assembled carotene plasmid was transferred into GFP-Ura yeast cells and verified by PCR and agarose gel electrophoresis. August 2 to August 9 The carotene plasmid and Delta-g plasmid were transferred into 4741 cells and passed verification. After testing the Delta-g plasmid to knock out the chromosomes of yeast cells, the carotene gene can still be expressed normally. Transfer the yeast containing Delta-g and carotene 4741 into the medium containing gal and the medium without gal, as the experimental group and the control group, let gal induce the expression of Delta-g and cut the chromosome. After 23 hours of incubation, the OD of the experimental group and the control group were measured and diluted by the same multiple (104, 103, 102) to plate the culture. Record the growth status of the cells, and record the number of colonies and the color of the colonies to verify that the carotene gene can still be expressed after the chromosome is absent. The results verified that the carotene gene did not appear in the cells, and the Delta-g band was normal. Due to the escape of the Delta-g plasmid, the carotene-Delta-g plasmid system was terminated. September 4 to September 23 The plasmid for extracting carotene is electrotransformed into Escherichia coli. The carotene plasmid was verified by PCR and agarose gel electrophoresis. After extracting the carotene plasmid, it was transferred to the chromosome-free yeast.
Construction of loxp-cre flip plasmid AUGUST 15 ~ AUGUST 22
August 15 to August 19 Construct loxp-Cre flip plasmid. August 20 to August 22 Transform the loxp-Cre plasmid into the yeast cell 4742 containing the 7flip plasmid to obtain the 7flip-Cre cell; The loxp-Cre plasmid was transformed into yeast cells 4742 and 4741 containing fGFP plasmid to obtain fGFP-Cre cells.
Construction of cutting plasmid
MAY 28 ~ SEPTEMBER 9We first constructed the Delta-g plasmid and verified the cutting efficiency, hoping that it can complete the construction of chromosome-free cells. However, in follow-up experiments, we found that the Delta-g plasmid is prone to escape, the Delta-g plasmid in the cell is very unstable, and gal itself is a carbohydrate substance, which is easily metabolized by the cell as an inducer, so we replaced it. The plasmid adopts 7flip-Cre and tetR-Cas9, which use estrogen as the inducer, which are more stable and difficult to escape, to complete the construction of chromosome-free cells.
Construct an empty plasmid
AUGUST 20 ~ AUGUST 30August 20 to August 23 An empty plasmid was constructed as a control group. The fragments were copied from the Delta-g plasmid by PCR and agarose gel electrophoresis for gel collection. The fragments were transformed into yeast cells 4741, and they were connected in the yeast cells. Part of the fragments were ligated using Overlap and then transferred into yeast cells 4741. Verify that it is fully connected by PCR and agarose gel electrophoresis. The empty plasmid is extracted from the yeast cells and electrotransformed into E. coli to complete the storage of the plasmid. August 24 to August 30 Transfer the extracted empty plasmid into yeast cells containing fGFP-Cre to obtain fGFP-empty-Cre cells; Each fragment was transformed into yeast cells containing fGFP-Cre, and they were connected in yeast cells. Connect some fragments using Overlap. Constructed a fGFP-empty-Cre plasmid that can realize a chromosome-free yeast cell control group.
Preparation of chromosome-free cells
Through the above experiments, we can see that we have constructed a control group of achromosomal yeast cells after the fGFP-empty-Cre plasmid is transferred into yeast cells 4742. We constructed an experimental group of chromosome-free yeast cells after the fGFP-7flip-Cre plasmid was transferred into yeast cells 4742-that is, all chromosomes were removed to complete the preparation of cell-free chromosomes. The experimental group and the control group were cultured in SC-L-H-U liquid medium for 8 to 10 hours at the same time, replaced with Sg-L-H-U medium and induced by adding estrogen, and then tested 48 hours later.
Form an efficiency display SEPTEMBER 7 ~ SEPTEMBER 24
Stream sorting: After the induction, the cells that do not contain green fluorescence are sorted out by flow cytometry to obtain chromosome-free cells.
Characterization of achromosome-free cells SEPTEMBER ~ OCTOBER
Verification of fGFP rapid degradation
Take the yeast 4742 containing fGFP as the experimental group, and the wild-type yeast 4742 as the control group for inoculation and culture, change the induction medium sg for 48 hours, change back to the normal medium SC, and continuously measure the fluorescence intensity of the two. Subtract the two fluorescence intensity to get the difference, which is the fluorescence signal value. If it is observed that the half-life of its fluorescence signal value is less than the half-life of gfp in the literature, it can indicate that fGFP is rapidly degraded.