Experimental Part

6.29-7.3

Purification of cas14 protein.

Open group meetings to discuss experimental details and familiarize with the experimental process of purified proteins

7.4

Purification of cas14 protein.

Prepare experimental materials for the purification of cas14 protein, strains, instruments, etc.

7.5

Purification of cas14 protein.

1.Preparation of LB medium 500ml*4

2. Shake the bacteria to recover.

Take LB medium 3ml*2 (TEV,MBP)

+Bacterial night 30 ul + antibiotic (Amp) ampicillin 3 ul

Constant temperature shaker 37℃ 180rpm 12-16he

7.6

Purification of cas14 protein.

1.Culture medium sterilization

2.Expansion, induction.

Take sterilized LB medium*4(TEV*2,MBP*2)

+500 ul antibiotic (Amp)+ 2ml ante-night bacteria night

Shaker 37℃ 180rpm 6-8h

Shake well for TEV and MBP

TEV+IPTG 50 ul * 237℃ 180 rpm for 12h

MBP+IPTG 100 ul * 218℃ 180 rpm for 12h

7.7

Purification of cas14 protein.

1.Bacteria-breaking lysis.

Take TEV and MBP and centrifuge at 10,000 rpm for 5 min, discard supernatant and addlysate 5 ml/tube (operate in ice box, move fast), blow well, and combine three tubes into onetube.

Crushing bacteria with ultrasonic 25% power on 4s off 10s working time 50-60mine

2.Preparation.

Purified water Equilibrium solution Elution solution (wash once)20% ethanol sequentialextraction on the machine, wash the machine (full water wash once) on the HS nickel column(1ml/min), wash the column, put the tube into the corresponding reagent bottle Syringe Filtermembrane filtration sample.

3.Purified proteins.

TEV and MBP maximum speed 5ml/min

A1 pure water A2 equilibrium solution B eluent A3 20% ethanol

When the machine is turned off, the water is washed once, and the unloading column(1ml\min) is washed once with ethano

4. Measure protein concentration (kit) Measure protein concentration (kit), 96-well plate, 100ul BCA working solution per well, 1ul Cu reagent 10ul protein, two protein two wells, measure absorbance, about 0.4 is normal value.

5.Mixed digestion with protein concentration 1:1 equal volume digestion (TEV slightly more) after mixing two tubes, 4℃ overnight

7.8

Purification of cas14 protein.

1.Protein purification

Ultrafiltration tube with filtered water, centrifuge 3800r 30min twice Equilibrium solution 30min twice

Top sample, centrifuge, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times on the machine.

Wash the machine with the same as before, wash the column to wash the equilibrium solution more than once, heparin column 1ml/min (pay special attention to sample evacuation)

7.9

Purification of cas14 protein.

Purified protein experimental results: purification failure, the experimental group met to discuss the reasons, may be operational errors, organize the material and re-purified cas14 protein.

7.13

Purification of cas14 protein.

1.Preparation of LB medium 500ml*4e'2.Shake the bacteria to recover.

Take LB medium 3ml*2 (TEV,MBP)

+Bacterial night 30 ul + antibiotic (Amp) ampicillin 3 ul

Constant temperature shaker 37℃ 180rpm 12-16h

7.14

Purification of cas14 protein.

1.Culture medium sterilization

2.Expansion, induction.

Take sterilized LB medium*4(TEV*2,MBP*2)

+500 ul antibiotic (Amp)+ 2ml ante-night bacteria night

Shaker 37℃ 180 rpm 6-8h

Shake well for TEV and MBP

TEV+IPTG 50 ul * 237℃ 180 rpm for 12h

MBP+IPTG 100 ul * 218℃ 180 rpm for 12h

7.15

Purification of cas14 protein.

1.Bacteria-breaking lysis.

Take TEV and MBP and centrifuge at 10,000 rpm for 5 min, discard supernatant and add lysis solution 5 ml/tube (operate in ice box, move fast), blow well, three tubes in one tube

Crushing bacteria with ultrasonic 25% power on 4s off 10s working time 50-60 min

2.Preparation.

Purified water Equilibrium solution Elution solution (wash once) 20% ethanol sequential extraction on the machine, wash the machine (full water wash once) on the HlS nickel column (1ml/min), wash the column, put the tube into the corresponding reagent bottle Syringe Filter membrane filtration sample

3.Purified proteins.

TEV and MBP maximum speed 5ml/min

A1 pure water A2 equilibrium solution B eluent A3 20% ethanol

When the machine is turned off, the water is washed once, and the unloading column (1ml\min) is washed once with ethanol

7.16

Purification of cas14 protein.

Measure protein concentration (kit) Measure protein concentration (kit), 96-well plate, 100ul BCA working solution per well, 1ul Cu reagent 10ul protein, two protein two wells, measure absorbance, about 0.4 is normal value

Mixed digestion with protein concentration 1:1 equal volume digestion(TEV slightly more) after mixing two tubes, overnight at 4℃

7.17

Purification of cas14 protein.

ultrafiltration tube, with filtered water, centrifuge 3800r 30mintwice Equilibrium solution 30min twice

Top sample, centrifuge, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times

Get on the machine

Wash the machine with the same as before, wash the column to wash the equilibrium solution more than once, heparin column 1ml/min (pay special attention to sample evacuation)

Problems: upper two tev lower two mbp: broken tube mix leads to experiment termination

7.18

Purification of cas14 protein.

The experiment was stopped due to an error, and members of the experimental group analyzed the experimental process to find the error

7.19

Purification of cas14 protein.

The experiment was stopped due to an error, and members of the experimental group analyzed the experimental process to find the error

7.20

Purification of cas14 protein.

After discussion, it was decided to change the eluent pH/elution column to re-purify cas14 protein.

7.21

Purification of cas14 protein.

1. Preparation of LB medium 500ml*4

2. Shake the bacteria to recover.

Take LB medium 3ml*2 (TEV,MBP)

+ bacterial night 30μl + antibiotic (Amp) ampicillin 3μl

Constant temperature shaker 37℃ 180rpm 12-16h

7.22

Purification of cas14 protein.

1. culture medium sterilization

2. Expansion, induction.

Take sterilized LB medium*4 (TEV*2,MBP*2)

+500μl of antibiotics (Amp) +2ml of ante-night bacteria night

Shake at 37℃ 180rpm for 6-8h

Shaken TEV and MBP

TEV+IPTG 50μl * 237℃ 180rpm 12h

MBP+IPTG 100μl * 218℃ 180rpm 12h

7.23

Purification of cas14 protein.

1. Bacteria-breaking lysis.

Take TEV, MBP high-speed centrifugation 10000 rpm 5min, discard the supernatant add lysate 5ml / tube (operation in the ice box, the action should be fast), blow well, three tubes in one tube

Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min

2. Preparation.

Purified water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, wash the machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the corresponding reagent bottle syringe filter membrane sample

3. Purification of proteins.

TEV and MBP max speed 5ml/min

A1 pure water A2 equilibrium solution B eluent A3 20% ethanol

When the machine is turned off, water wash once, unload the column (1ml/min) ethanol wash once

Figure 2. Computer display when performing protein purification experiments

4. measurement of protein concentration (kit) measurement of protein concentration (kit), 96-well plate, each well, 100ul BCA working solution, 1ul Cu reagent 10ul protein, two protein two wells, measurement of absorbance, 0.4 or so is the normal value

Figure 4. Computer display when performing protein purification experiments

5. Mixed digestion with protein concentration 1:1 equal volume digestion (TEV slightly more) after mixing two tubes, 4℃ overnight

7.24

Purification of cas14 protein.

1. Protein purification

Ultrafilter tube with filtered water, centrifuge 3800r 30min twice Equilibrium solution 30min twice

Top sample, centrifugation, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times

Loading the machine

Wash the machine as before, wash the column to wash the equilibrium solution once more, heparin column 1ml/min (pay special attention to sample evacuation)

7.25

Purification of cas14 protein.

After discussion and testing, the strain was suspected to be improperly preserved, and the failed experience was summarized to optimize the experimental process

7.26

Purification of cas14 protein.

Repurify cas14 protein

7.27

Purification of cas14 protein.

Repurify cas14 protein

7.28

Purification of cas14 protein.

Repurify cas14 protein

7.29

Purification of cas14 protein.

Repurify cas14 protein

7.30

Purified cas14 protein.

Protein purification successfully, continue to determine mbp, tev

7.31

Purified cas14 protein.

The assay failed, the experiment held a group meeting to discuss, further analyze the reasons and optimize the experimental process

8.6

Purification of cas14 protein.

Review the experimental procedure and find the possible reasons why mbp and tev could not be measured: eluent ph is too high/bacterial problems/column problems, etc.

8.7

Purified cas14 protein.

Repurification of cas14 protein

8.8

Purification of cas14 protein.

Repurify cas14 protein

8.9

Purified cas14 protein.

Repurify cas14 protein

8.10

Purification of cas14 protein.

Repurify cas14 protein

8.11

Purification of cas14 protein.

Successful cas14 protein purification

8.13

Purification of csm6 protein.

8.14

Purification of csm6 protein.

1. Preparation of LB medium 500ml*2 Sterilization

2. Shake the bacteria to recover.

Take LB medium 3ml*2

+ 30μl of bacterial night + 3μl of caramycin

Constant temperature shaker 37℃ 180rpm 12-16h

8.15

Purification of csm6 protein.

1. Expansion, induction.

Take sterilized LB medium + 500μl caramycin + 3ml antecedent bacterial night

Shake 37℃ 180rpm 6-8h

Shake the good bacteria solution

Csm6+IPTG 100μl 18℃ 180rpm 12h

8.16

Purification of csm6 protein.

1. Bacterial lysis by breaking.

Take the bacteriophage solution high-speed centrifugation 10000rps 5min, discard the supernatant add lysate, blow evenly, three tubes in one tube

Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min

2. Preparation.

Pure water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, cleaning machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the corresponding reagent bottle syringe filter membrane sample

8.17

Purification of csm6 protein.

Run Gel Validation

SDS-page protein gel electrophoresis

1. gel preparation

2. sample processing

3. add maker and sample

4. add running buffer and perform electrophoresis according to the procedure

5. stain the plate and decolorize

6. observation

Figure 8. Running glue results are displayed under the machine

8.18

Purification of csm6 protein.

Summarize the experimental results csm6 protein purification successfully

8.24

Preparing for rna extraction

8.25

Extraction of RNA.

21:00 200ml LB liquid medium, 1.5ml EP tube, 10ml EP tube sterilized

8.26

Extraction of RNA.

9:00 Take 5ml liquid medium + 200ul bacteria and shake it.

9:20 Mix lysozyme solution with TE buffer to a concentration of 15mg/ml, mix, dispense appropriate amount and store at -20℃.Dilute RNA wash buffer II with 100% alcohol (alcohol:buffer2=4:1, store at room temperature)

15:00 Extraction of total RNA using E.Z.N.A. Bacterial RNA Spin kit

(Refer to the kit instructions for the experimental procedure)

The obtained RNA samples are stored at -80°C

17:40 Preparation of TAEbuffer for gum preparation

Run gum 2% TAE agarose condensate 5ul of sample

8.27

Extraction of RNA.

21:00 100ml LB liquid medium, 10ml,1.5ml EP tube sterilization

9:00 5ml LB liquid medium+200ul bacteria reagents shake bacteria

9:20 500ml LB solid medium configuration, sterilization

11:40 Add antibiotics to the solid medium, pour the plate and put it into 4℃ refrigerator for backup

15：00 Dilute the bacterial solution by the following multiples

10	10²	10³	10⁴	10⁵	10⁶	10⁷	10⁸	10⁹

Measure the OD value of undiluted bacterial solution, diluted bacterial solution; medium is also diluted and measure the OD value

Figure 11. Experimental results under different conditions

16：30 Select 10⁴, 10⁶, 10⁸ dilutions of bacterial solution to coat the plate, add 100ul of bacterial solution to each plate

19:00 Put the coated plates into 37℃ incubator and count the bacteria the next day.

8.29

Extraction of RNA.

10:00 Counting bacteria

8.30

Extraction of RNA.

21:00 200ml LB liquid medium, 1.5ml EP tubes, 10ml EP tubes sterilized

8.31

Extraction of RNA.

Take 5ml of liquid medium + 200ul of bacterium and shake the bacterium.

Mix lysozyme solution with TE buffer to a concentration of 15mg/ml, mix in, dispense the appropriate amount and store at -20°C

Dilute RNA wash buffer II with 100% alcohol (alcohol:buffer2=4:1, store at room temperature) Extract total RNA using E.Z.N.A. Bacterial RNA Spin kit

The obtained RNA samples were stored at -80°C

17:40 Preparation of TAEbuffer Gum preparation

Run gum 2% TAE agarose condensate 5ul of sample

Mutual aid experiment: the virulence gene plasmid arrives, start preparing for mutual aid experiment

9.1

Extraction of RNA.

Prepare 100ml LB liquid medium, 10ml,1.5ml EP tube sterilization

Mutual experiment: learn and communicate the experimental procedure Prepare experimental materials

9.2

Extraction of RNA.

LB liquid medium + 200ul bacteria Resuscitation shaking bacteria

500ml LB solid medium configuration, sterilization

Add antibiotics to the solid medium, pour the plate and put it into 4℃ refrigerator for backup

Dilute the bacterial solution by the following multiples

10	10²	10³	10⁴	10⁵	10⁶	10⁷	10⁸	10⁹

Measure the OD value of undiluted bacterial solution, diluted bacterial solution; the same dilution of the medium and measure the OD value

OD value of 0.444 for one group and 0.241 for one group

OD value 0.444 group choose to dilute 10⁶, 10⁷, 10⁸ times to coat the plate

OD value 0.241 group choose to dilute 10, 10², 10³ times to coat the plate

Figure 15. Results under different experimental conditions

9.3

Extraction of RNA.

Counting bacteria

9.4

Mutual Aid Experiment.

Learning and Communicating Experimental Procedures Preparing Experimental Materials

9.5

Mutual Aid Experiment.

9.6

Mutual Aid Experiment.

9.8

Reciprocal experiment.

9.9

Extraction of RNA.

200 ml LB liquid medium, 1.5 ml EP tubes, 10 ml EP tubes sterilized

9.10

Extraction of RNA.

Take 5ml of liquid medium + 200ul of bacteria and shake the bacteria.

Lysozyme solution and TE buffer mixed to a concentration of 15mg/ml, mixed in, divided into appropriate amounts, stored at -20 ℃

Dilute RNA wash buffer II with 100% alcohol (alcohol:buffer2=4:1, store at room temperature)

Extract total RNA using E.Z.N.A. Bacterial RNA Spin kit

The obtained RNA samples were stored at -80°C

17:40 Preparation of TAEbuffer Gum preparation

Run gum 2% TAE agarose condensate on sample 5ul

Reciprocal experiment.

Amplification of 4 pairs of primers for virulence genes pcr attempt with medium (solid liquid) Sterilization Gum preparation and running gum Plate scribing and running gum

9.11

Extraction of RNA.

Prepare 100 ml LB liquid medium, 10 ml,1.5 ml EP tubes sterilized

Reciprocal experiments.

PcR amplification (same day as PCR - the previous day is pre-experiment) But x 4 total 16) Glue making and glue running Glue sterilization for eight consecutive tubes, gun tip

Figure 22. Colonies of culture medium

Figure 23. Extraction of pro plasmids

Figure 24. blra rubber recycling

9.12

Extraction of RNA.

LB liquid medium + 200ul bacteria Resuscitation shaking bacteria

500ml LB solid medium configuration, sterilization

Add antibiotics to the solid medium, pour the plate and put it into 4℃ refrigerator for backup

Dilute the bacterial solution by the following multiples

10	10²	10³	10⁴	10⁵	10⁶	10⁷	10⁸	10⁹

Measure the OD value of undiluted bacterial solution, diluted bacterial solution; the same dilution of the medium and measure the OD value

One group 0.488, one group 0.157

OD value 0.488 group choose to dilute 10⁴, 10⁵, 10⁶ times coated plate

OD value 0.157 group choose to dilute 10³, 10⁴, 10⁵ times to coat the plate

pro plasmid try pcr amplification (pre-experiment - probe concentration + try parameters)

9.13

Extraction of RNA.

Counting bacteria

Reciprocal experiments.

Results are with heterobands all selective gel recovery, no selective column recovery. -- Do more tubes of amplification the next day.

No band OR with trailing band: fragment is too long, template concentration is too high, annealing temperature is too low, primer specificity is not good

Figure 28. The instruments we use for experiments

9.14

Mutual aid experiment.

Failure reason analysis.

1. added the primer of the connector may be a little less primer specificity

2. yesterday's pcr template 1 overnight degradation

3. plasmid did not mention well: too much bacteriophage, plasmid may be mixed with a lot of broken E. coli genomic DNA

Correct the error and re-experiment

9.15

Reciprocal experiment.

Extraction of plasmids with arabinose antibiotics

9.16

Mutual aid experiment.

Pre-amplification of pcr

Results: abnormal but with bands

Figure 30. Display of the results of the glue run on the computer

9.17

Reciprocal experiment.

Fold amplification Recovery

Result: Failure Error during glue run

Figure 31. Gum recovery results of mishandling

9.18

Reciprocal experiment.

Repeat 9.17 + Method II Method III for double digestion

9.19

Reciprocal experiment.

Transformation Homologous recombination (method 3, method 4) Double digestion (method 1)

Results: method 1 needs to be redone Adding the wrong enzyme

9.20

Reciprocal experiment.

Observation plate: no bacteria growth Suspect transformation problem

PCR with recovery to do 1step of pro

Make solid medium + sterilize the gun tip and eight-link tube

9.22

Mutual aid experiment.

Discuss and analyze the possible causes with the mutual help subjects.

1. not completely protected from light, toxic genes in the natural light leakage

2. Failure due to inaccurate heating and time

3. plasmid base number is large, self-spitting phenomenon occurs, need to keep changing the sensory state to try

4. reverse pcr, pro plasmid is too long and dragging, etc.

5. The enzyme used is not common

9.23

Mutual help experiment.

Summarize the reasons for failure and then discuss the procedure of subsequent experiments

9.24

Reciprocal experiment.

E. coli receptor state, 1 μL of virulence gene plasmid was added to 50 μL of receptor state, plates were coated after transformation according to the transformation process, 50 μL of solution was added to each plate and placed in 30°C for overnight incubation.

9.25

Extraction of RNA.

9：00 Resuscitation shake (5ml LB medium + 200ul bacteriophage solution)

14：00 Measure OD bacteriophage value, dilute the bacteriophage to about 0.157 with medium

14:30 Dilute the bacterial solution to 0.127 with sterile water 10⁵ times

14:40 Total RNA was extracted simultaneously from the original and 10⁵ times diluted bacterial broth using the E.Z.N.A. Bacterial RNA Spin kit.

(Refer to the kit instructions for the experimental procedure)

The obtained RNA samples were stored at -80°C

Reciprocal experiments.

9.27

Extraction of RNA.

11:00 Run nucleic acid gel

Prepare 2% TAE agarose agglutination solution

Sample 5ul

Failure, the strips did not run, probably because the TAE solution has been prepared for too long and has deteriorated