NOTEBOOK

1. pYB1a-PobR-eGFP-Cmr constructing experiment.
2. Preliminary experiments of chloramphenicol screening.

Check out our daily lab transcript here→

1. Optimization of induction conditions: 20 mg/mL chloramphenicol different concentrations of 4HB (Three groups of repeats); Changes of fluorescence values in different 4HB concentrations in 8.5 hour.
2. Screening of random mutation: Re-screening validation data of 16-F4 and 18-B4.
3. Re-screening: T5 plate.

Check out our daily lab transcript here→

1. HMA gradient verification: 16-F4.
2. Sequencing: 16-F4 and 18-B4.

Check out our daily lab transcript here→

1. Replace the ori of vector: We used Gibson ligation and electrophoresis detection for the bacteria replaced by the hmaS vector and hmaS-tktA-ppsA vector respectively after PCR.

Check out our daily lab transcript here→

1. Error analysis of 96-well plates and deep-hole plates.

Check out our daily lab transcript here→

1. Replace the 4HB solvent: Anhydrous ethanol was used to reconfigure 4HB for the solution, and the difference between it and methanol in response to 4HB was verified.

Check out our daily lab transcript here→

1. Correction formula.

Check out our daily lab transcript here→

1. Determination of engineering bacteria by HPLC.
2. Protein expression of engineering bacteria.
3. Response condition groping:
   (1) Test the toxicity of 4HB and HMA.
   (2) Find the appropriate concentration of 4HB.
   (3) HMA validation of control bacteria.
4. Cmr gradient experiment.
5. Exploration of first screening on solid medium.

Check out our daily lab transcript here→

1. Determination of engineering bacteria by HPLC.
2. Screening.
3. Liquid initial screening conditions.
4. Re-screening verification.
5. First screening of solid medium conditions.

Check out our daily lab transcript here→

1. Re-screening verification: no strains that meet the requirements have been screened out.
2. Megahop PCR: Perform error-prone PCR on pYB1a-PobR-eGFP-Cmr, and then purify and recover the PCR product.
3. Preliminary test of flat plate screening: to determine whether the low-intensity induction can make the strain grow normally under the condition of adding 0.1% 20 mg/mL and 30 mg/mL chloramphenicol.

Check out our daily lab transcript here→

1. Re-screening verification: A total of 21 strains were re-screened and verified. There are no strains that meet the standards and the labeled strains have been sent for sequencing.
2. Preliminary experiment of flat plate screening: The plate screening feasibility experiment was carried out, and the BW control bacteria (only responding to 4HB) were quantified and plated. Then, estimate the mutation library capacity.
3. Megahop PCR system modification: We modified the Megahop PCR system for constructing pYB1a-pobR-eGFP-Cmr.

Check out our daily lab transcript here→

1. Construction of engineering bacteria.
2. Perform PCR product: A total of 6 bacteria were sent for re-screening this week.
3. Re-screening verification: No strains that meet the requirements appeared.
4. Preparation of electrocompetence.

Check out our daily lab transcript here→

1. Photocopying.
2. Re-screening verification.
3. Verification of the efficiency of commercialization to competence.
4. Construction of engineering bacteria.

Check out our daily lab transcript here→

1. Plate screening.
2. Re-screening.
3. pTarget-aspC Construction.

Check out our daily lab transcript here→

1. Engineering bacteria.
2. Photocopying.
3. Re-screening.
4. Re-screening verification.
5. Data collation.

Check out our daily lab transcript here→

1. Re-screening.
2. Re-screening verification.
3. Verification of possible strains.
4. Conversion coating.
5. Re-screening.

Check out our daily lab transcript here→

1. Engineering bacteria.
2. Changes in the calculation method of induction strength.
3. Re-screening.
4. Re-screening verification.
5. HMA Gradient Verification.
6. HMA analog response verification. New HMA analog response verification.

Check out our daily lab transcript here→

1. Changes in the calculation method of induction strength.
2. Re-processing of re-screening data.
3. Re-screening verification.
4. HMA gradient verification.
5. Verification of HMA analog response.
6. Sequencing.

Check out our daily lab transcript here→

1. Gene knockout: knockout tyrR on the bases of BL21 .
2. HMA ligand specificity test: Have HMA ligand specificity test for the preliminarily screened biosensors and DH5αpobRWT.
3. Fluorescence re-screening.
4. Site-directed mutagenesis of the PobR CDS.

Check out our daily lab transcript here→

1. Site-directed mutagenesis of the PobR CDS.
2. Gene knockout: We removed pTarget-tyrB from the strain. Then, we tried to knock tyrR out based on BL21ΔtyrB.
3. Site-directed mutagenesis of the PobR CDS.
4. Modeling and docking.
5. Re-screening: We stabilized the system and screened the 647 clones selected last time, then we re-screened the clones (83 clones in total).
6. Chloramphenicol ALE pre-experiment.

Check out our daily lab transcript here→

1. Gene knockout: tyrR knockout; aspC knockout; sequencing of pTarget-aspC.
2. Gradient HMA concentration induction assay & fluorescence re-screening: F5-B7.
3. Ligand specificity test: F7-E8-3, F5-A10-1, F6-F4-3, F5-B7-2 and DH5αpobRWT.
4. Modeling and docking: Docking between structure by RoseTTAFold and 4HB; docking between structure by AlphaFold2 and 4HB; the PobR protein structure was simulated by RoseTTAFold; the protein structure of PobR was simulated using AlphaFold2, and AlphaFold2 was run through cloud computing platform; comparison of RoseTTAFold and AlphaFold2.
5. Site-directed mutagenesis of the PobR CDS.
6. Chloramphenicol ALE pre-experiment.

Check out our daily lab transcript here→

1. Site-directed mutagenesis of the PobR CDS.
2. Gene knockout: aspC knockout.
3. Gradient HMA & 4HB concentration induction assay.
4. Chloramphenicol ALE pre-experiment.
5. Ligand specificity test: F7-E8-3, F6-F4-3 and DH5αpobRWT.

Check out our daily lab transcript here→

1. Gene knockout: aspC knockout
2. Chloramphenicol ALE pre-experiment.
3. Fluorescence re-screening of single amino acid mutants.

Check out our daily lab transcript here→

1. pYB1a-PobR^mut(F5-B7)-eGFP-SacB constructing experiment.
2. pYB1a-PobR^mut(F5-B7)-eGFP-SacB sucrose gradient pretest.
3. pRB1s-hmaS generation of mutant library replacing with Anderson promoter.

Check out our daily lab transcript here→

1. HMA-0 constructing experiment.
2. Adaptive evolution experiment under chloramphenicol pressure.

Check out our daily lab transcript here→

1. HMA-0 constructing experiment: We double transformed pRB1s-HmaS-aroG- pheA, pLT1k-MP6 and pYB1a- PobR^mut(F5B7)- eGFP- Cmr into BL21 and BW.
2. Adaptive evolution experiment under chloramphenicol pressure: HMA-C60-1, HMA-C60-2, HMA-C65-2, HMA-C70-1 and HMA-C75-1.

Check out our daily lab transcript here→

1. Adaptive evolution experiment under chloramphenicol pressure: HMA-C80-1, HMA-0, HMA-50,        HMA-60-1, HMA-60-2, HMA-65-1, HMA-65-2, HMA-70-1, HMA-75-1 and HMA-80-1.

Check out our daily lab transcript here→

Click a item below to view the relevant experiment record.

1.Characterization of ligand responses of PobR^WT.

2.Design and construction of the PobR mutant library.

3.Screening for HMA-responsive PobR mutants.

4.Ligand specificity and detection ranges of the two selected PobR mutants.

5.Functional evaluation of individual mutations of identified amino acids in PobR.

6.Modeling and docking.

7.HMA-responsive sensor-driven adaptive laboratory evolution (ALE).