2024.3-2024.5

Week1 3.2-3.9

Characterization plasmids pCL-100RFP, pCL-110RFP, pCL-116RFP,pCL-109RFP, pCL-113RFP and pCL-103RFP were constructed by fusing the reporter genes rfp and J23100/J23110/J23116-B0034 or J23103/J23113/J23109-B0033 to plasmid pCL1920 by Gibson seamless DNA cloning.

We introduced these plasmids into DGF298 by electroporation.

The strengths of J23103/J23113/J23109-RBS33 and J23116/J23110/J23100-RBS34 expression elements at DGF298 were shown by the RFP fluorescence intensity.

Week2 3.9-3.16

The E. coli strain DGF298-PTK-Red containing Homologous recombination system was obtained and preserved in a glycerol tube.

Competent cells of DGF298 were constructed and the plasmid PTK-Red was transformed by electroporation. Then the colonies containing PTK-Red were selected by Spectinomycin resistance screening.

After PCR and gel electrophoresis validation we obtained the strain DGF298-PTK-Red. The plasmid was sequenced as well.

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Figure 1 Map of plasmid PTK-Red

Figure 2 Gel electrophoresis results of PTK-Red

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Week3 3.16-3.23

The DNA fragment ftsZ-Kan was obtained, purified and phosphorylated.

Gene fragments JAR3-LR and FtsZ-Kan was obtained by PCR based on plasmids stored in the laboratory.

The plasmid JAR3-LR-ftsZ-Kan was assembled by Gibson Assembly and transferred into DH5α. At the same time, DH5α containing the plasmid was selected by Kanamycin resistance screening.

The gene fragment ftsZ-Kan was obtained by PCR and sequenced.

The fragment was purified and phosphorylated by kits respectively.

The fragment was verified by gel electrophoresis, then recycled by Agarose gel DNA recovery kit .

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Figure 3 DNA Fragment ftsZ-Kan

Figure 4 Gel electrophoresis results of ftsZ-Kan

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Week4 3.23-3.30

The gene fragment ftsZ-Kan was transferred into the strain DGF298-PTK-Red by electroporation.

At the same time, the gene fragment ftsZ-Kan was inserted into the genome of DGF-298, and the polyploid minimal chassis cell DGF298-103Z was constructed. Polyploid colonies were selected by Kanamycin resistance screening.

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Figure 5 Fragment insertion sites and validation primers on the DGF-298 genome

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Week5 3.30-4.6

The ploidy of DGF298-103Z was verified by PCR and gel electrophoresis, the result showed that the presence of wild-type chromosomes and engineered chromosomes.

The fragment ftsZ-Kan in the strain was sequenced at the same time.

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Figure 6 Gel electrophoresis results of DGF298-103Z

Figure 7 Sequencing results of ftsZ-Kan

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Week6 4.6-4.13

The morphological changes of Escherichia coli DGF298-103Z from DGF298 was verified by oil microscope view and field emission scanning electron microscopy（SEM）respectively.

Week7 4.13-4.20

In order to further verify the changes in the DGF298-103Z chromosome, DGF298 and DGF298-103Z were subjected to DAPI staining and flow cytometry observation.

Week8 4.20-4.27

To verify the chromosomal genetic stability of polyploid E. coli DGF298-103Z, the strain was transferred 6 times in a row in shake flask medium. After each transfer, we isolated and picked colonies for PCR and gel electrophoresis validation, which showed that the polyploid strain DGF298-103Z was highly stable. From this, we proposed the concept of PMEC (Minimalist polyploid Escherichia coli).

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Figure 8 After the 5th and 6th transitions, single colonies will grow

Figure 9 Gel electrophoresis results after transfer

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Week9 4.27-5.4

We performed robust phenotypic analysis of strains DGF298 and DGF298-103Z by measuring the growth density of cells and the mass doubling time of the exponential phase under the conditions of LB medium and M9 medium of different pH, acetate concentration, temperature. The first is the analysis of LB medium at different pH and M9 culture conditions. Three replicates of single colonies were performed in each group.

Table 1 Robustness analysis of DGF298-103Z and DGF298 under different conditions of pH and mediums.//caption

Strains	Mediums	pH	Method
DGF298-103Z	LB	5	Measure the growth density of cells and the mass doubling time with microplate reader
		6
		7
	M9	5
		6
		7
DGF298	LB	5
		6
		7
	M9	5
		6
		7

Week10 5.4-5.11

We performed robust phenotypic analysis of strains DGF298 and DGF298-103Z by measuring the growth density of cells and the mass doubling time of the exponential phase under the conditions of LB medium and M9 medium of different acetate concentration. Three replicates of single colonies were performed in each group.

Table 2 Robustness analysis of DGF298-103Z and DGF298 under different conditions of acetate concentration and mediums.//caption

Strains	Mediums	Acetate concentration(g/L)	Method
DGF298-103Z	LB	0	Measure the growth density of cells and the mass doubling time with microplate reader
		1
		2
		4
	M9	0
		1
		2
		4
DGF298	LB	0
		1
		2
		4
	M9	0
		1
		2
		4

Week11 5.11-5.18

We performed robust phenotypic analysis of strains DGF298 and DGF298-103Z by measuring the growth density of cells and the mass doubling time of the exponential phase under the conditions of LB medium and M9 medium of different temperature. Three replicates of single colonies were performed in each group.

Table 3 Robustness analysis of DGF298-103Z and DGF298 under different conditions of temperature and mediums.//caption

Strains	Mediums	Temperature (℃)	Method
DGF298-103Z	LB	30	Measure the growth density of cells and the mass doubling time with microplate reader
		37
		42
		45
	M9	30
		37
		42
		45
DGF298	LB	30
		37
		42
		45
	M9	30
		37
		42
		45

2024.5-2024.7

Week12 5.18-5.25

In order to explore the changes of protein expression and carbon flux in the polyploidized strain DGF298-103Z, we decided to perform 16s rRNA sequencing identification analysis and transcription library analysis of polyploid E. coli DGF298-103Z and haploid E. coli DGF298.

We prepared DNA from the samples and identified the purity of the sample species by 16sRNA sequencing.

Week13 5.25-6.1

We performed a second-generation of high-throughput transcriptome sequencing of the samples.

Week14 6.1-6.8

We analyzed the measured transcriptome data, including metabolic pathway gene expression level, GC content, COG classification annotation, KEGG classification annotation, SSR. We found that the expression levels of carboxylic acid synthesis pathways, amino acid synthesis pathways, and sulfur compound synthesis pathways of DGF298-103 were upregulated in polyploids.

Week15 6.8-6.15

By analyzing the transcriptome sequencing results, we found that the expression levels of polyploid E. coli DGF298-103Z were up-regulated in the carboxylic acid synthesis pathway, amino acid synthesis pathway, and sulfuric acid compound synthesis pathway. To quantify the effect of differentially expressed genes on metabolic flux at the genome scale, we established a genome-level metabolic model of DGF-298 and its polyploidy. After substituting the transcriptome data, we found that the down-regulation of two genes reduced the biomass response rate of bacteria, i.e., pfkB and nuoF, so we decided to upregulate these two genes by a combination of promoters and RBS of different intensities to see how they affected the growth of the strain. High intensity combinations were named U, medium intensity combinations M, and low intensity combination is named as the D.

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Figure 10 Modular control of different promoter and RBS combinations

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Week16 6.15-6.22

We designed primers at both ends of nuoF and pfkB and performed two rounds of PCR separately. After each round of PCR, we separated these fragments by agarose gel electrophoresis and recovered them through the kit. These fragments are then used for Gibson assembly with plasmid backbones. We chemically transformed the assembled plasmids into the strain DH5α for storage and amplification. Then we validate these plasmids by PCR and sequencing.

Week17 6.22-6.29

Since all plasmids except the plasmids DU and DM had varying degrees of fragment deletion and point mutations, we reconstructed them.

We successfully reconstructed plasmids UD and MU. An error occurred with the other plasmids.

We extracted the plasmid that was successfully constructed.

Week18 6.29-7.6

We transferred the validated plasmids UD, MU, DU, DM into the strain DGF298-103Z by electroporation. Similarly, PCR and sequencing confirmed that these plasmids were unmistakably expressed at DGF298-103Z.

At the same time, we reconstructed the remaining plasmids: UU, UM, MM, MD. Sequencing results showed that they all underwent varying degrees of electrical mutations. Through communication with the mentor, the cause of the plasmid mutation may be due to the high expression of the protein that may be putting metabolic stress on the strain.

Week19 7.6-7.13

We performed RT-qPCR experiments to see the expression levels of the nuoF and pfkB genes of DGF298-103Z strains that have been transferred into the regulatory plasmid.

Week20 7.13-7.20

We characterized the growth of polyploid strains with varying degrees of upregulation of nuoF and pfkB in the experimental group and the haploid strain DGF298 and polyploid strain DGF298-103Z in the control group under LB culture conditions with a microplate reader. We found that the logarithmic phase of some experimental strains was earlier than that of DGF298-103Z.

2024.7-2024.9

Week21 7.20-7.27

In transcriptome analysis, we found that polyploid E. coli may have a better fermentation potential. Through communication with the mentors, we decided to select the appropriate vectors, construct different heterologous gene plasmids and introduce them into the cells to analyze their production capacity for different high value-added products. The first is to explore the heterologous protein expression potential of polyploid E. coli by expressing GFP, the other is to explore the potential of PHB fermentation production by expressing PHB production genes.

Week22 7.27-8.3

The plasmid PACYC-Ptac-GFP-Amp was assembled by Gibson method using the backbone fragment PACYC-Ptac-GFP and the Ampicillin resistance gene fragment the laboratory already has.

Then the plasmid was transferred into DH5α. After that, the plasmid was verified by gel electrophoresis. It was sequenced at the same time.

The plasmid was extracted the next day.

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Figure 11 Map of plasmid PACYC-Ptac-GFP-Amp

Figure 12 Sequencing results of plasmid PACYC-Ptac-GFP-Amp

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Week23 8.3-8.10

Competent cells of DGF298 and DGF298-103Z were induced and the obtained plasmid PACYC-Ptac-GFP-Amp was transformed into E.coli strain DGF298 and DGF298-103Z by electroporation.

Then the colonies containing PACYC-Ptac-GFP-Amp were selected by Ampicillin resistance screening. At the same time, the engineered chromosome of DGF298-103Z was sustained by Kanamycin resistance.

After PCR and gel electrophoresis validation we obtained the strains DGF298-GFP and DGF298-103Z-GFP.

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Figure 13 Gel electrophoresis results

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Next, the expression levels of green fluorescent protein (GFP) in DGF298 and DGF298-103Z under LB liquid medium and M9 medium conditions were detected by microplate reader. We also observed the fluorescence intensity of colonies of DGF298-GFP and DGF298-103Z-GFP by blue light detector.

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Figure 14 Observing the fluorescence intensity of colonies of DGF298-GFP(left) and DGF298-103Z-GFP(right) by blue light detector

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Week24 8.10-8.17

E.coli DH5α containing plasmid Puc-Pcon-PHB were obtained and preserved in a glycerol tube.

The plasmid was extracted the next day.

After that, the plasmid was transformed into DGF298, DGF298-103Z and W3110 by electroporation.

Colonies containing the plasmid were selected by Ampicillin resistance screening.

We verified the strains by gel electrophoresis and sequencing.

The results shows that we obtained the strains DGF298-PHB, DGF298-103Z-PHB and W3110-PHB.

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Figure 15 Map of plasmid pUC-Pcon-PHB

Figure 16 Results of gel electrophoresis

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Week25 8.17-8.24

Given the controllability of M9 medium, we selected M9 as the fermentation medium. The strains DGF298-PHB, DGF298-103Z-PHB and W3110-PHB were fermented in shake flasks to simulate fermentation, and the cell growth level OD600 and sugar consumption level were measured during the fermentation process.

Colonies of the strains containing Puc-Pcon-PHB were isolated during the fermentation to test the production of poly-β-hydroxybutyrate (PHB) by Nile red staining.

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Figure 17 Staining results of Nile red at the late stage of fermentation (DGF298-PHB, W3110-PHB and DGF298-103Z-PHB)

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Week26 8.24-8.31

In order to explore whether DGF298-103Z has advantages in amino acid fermentation, we analyzed the composition of free amino acids in DGF298, DGF298-103Z and W3110 shake flask fermentation broths by liquid chromatography.

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Table 4 Amino acid content

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At the same time, the contents of PHB in DGF298, DGF298-103Z and W3110 bacteria were detected by gas chromatography after the fermentation in shake flasks.

Table 5 Dry weight of the cell and PHB content of the sample//caption

Name	Peak area	PHB concentration in gas phase vials (g/L)	Peak time(min)	Bacterial dry weight (mg)	Total cell dry weight (mg)	PHB concentration in shake flask (g/L)
DGF298	127491.1	4.1063633	3.043	0.13	21	0.013267
DGF298	134318.9	4.3111487	3.004	0.12	20.8	0.014945
DGF298	134819.1	4.3261793	3.035	0.09	16.2	0.015574
DGF298-103Z	144921.1	4.6292333	3.09	0.04	12.3	0.02847
DGF298-103Z	154920.3	4.9292039	3.01	0.07	20.9	0.029434
DGF298-103Z	143312.4	4.5809642	3.043	0.09	18.2	0.018527
W3110	113034.7	3.6726491	3.036	0.12	20.1	0.012303
W3110	110441.9	3.5948507	3.035	0.11	23.4	0.015294
W3110	81124.8	2.7153434	3.049	0.11	25.5	0.012589

Week27 8.31-9.7

To explore the potential sensitivity of Corynebacterium glutamicum’s shuttle promoter to specific metabolic conditions and its transferability to E. coli DH5α, we introduced various shuttle promoters into E. coli DH5α and screened for robust expression. Subsequently, these promoters’ sequences were isolated from the genome and cloned upstream of the reporter gene rfp. These constructs were then utilized to evaluate the expression levels of different constitutive promoters under controlled conditions. We fused reporter genes rfp and P002/P009/P012/P022/P026/P038/P039/P040/P043/P048, assembling them into the plasmid PACYAC using seamless DNA cloning, resulting in characterisation plasmids PACYAC-002RFP, PACYAC-009RFP, PACYAC-012RFP, PACYAC-022RFP, PACYAC-026RFP, PACYAC-038RFP, PACYAC-039RFP, PACYAC-040RFP, PACYAC-043RFP, and PACYAC-0048RFP.”

Week28 9.07-9.14

We electrotransformed different plasmids into DGF-298 and characterized them. Our experimental conditions for characterizing this part were as follows:

• Plasmid Backbone: PACYC
• Chassis cell ：E. coli DH5α
• Medium: 1.5 mL of liquid LB medium
• Condition 37°C, 24 h, under vigorous shaking
• Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.)

Fluorescence intensity was detected using an enzyme marker at an excitation wavelength of 590 nm and an emission wavelength of 645 nm, and fluorescence intensity values (a.u.) were obtained by calculating the fluorescence intensity to OD₆₀₀ ratio.

Week29 9.14-9.21

We initiated the assembly of self-induced dynamic time-sequence modulated cascade systems. The foundational plasmids, pCDF-QS-GFP and Plas-RFP, were graciously provided by Xiaomeng Li under the supervision of Prof. Quanfeng Liang. Utilizing pCDF-QS-GFP as the basis for plasmid construction, we conducted PCR amplification to generate Fragment 1 with primers D-LasR-F and LasI-P02-R, and Fragment 2 with primers D-LasR-R and LasI-P02-F. These fragments were merged via Gibson assembly and introduced into the DGF-298 receptor through chemical transformation. The resultant plasmid QS-P02-LasI-GFP was obtained. Subsequent iterations involved adapting the primer sequences to accommodate various promoters, such as P38, and following analogous assembly procedures. Through co-transfection of the engineered plasmids with Plas-RFP into the chemoreceptor state of DGF-298, new strains denoted L02 and L38 were generated.

Week30 9.21-9.28

We characterized the plasmids that have been constructed so far, protocal as follows:

• Plasmid Backbone: pCDF(for GFP), pUC（for RFP）
• Chassis cell ：E. coli DGF-298
• Medium: 1.5 mL of liquid LB medium
• Condition 37°C, 24 h, under vigorous shaking
• Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.)

The fluorescence intensity of RFP was detected using an enzyme marker with an excitation wavelength of 558 nm and an emission wavelength of 583 nm, and that of GFP was detected using an enzyme marker with an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and the values of the fluorescence intensity were obtained by calculating the ratio of the fluorescence intensity to the OD600 (a.u.).