Part:BBa_K5343016
J23109-RBS(BBa_B0033)-GFP
Usage and Biology
Application of Cobrapy in GSSM modelling of polyploid Escherichia coli:
The main work of our experimental group started with E. coli DGF-298 and the construction of its prokaryotic polyploid E. coli DGF-298-103Z. DGF-298 is an extremely parsimonious strain with a streamlined genome. The main goal of the first part of the modelling was to model the metabolic network of DGF-298 as well as its polyploid and to analyse the relationship between various factors and PHB production as well as the effect of polyploidisation on metabolic flow. Conventional means such as chemical reaction kinetics modelling are limited by how well we know the various reaction parameters and by the size of the metabolic network, making it difficult to simulate metabolic flow on a larger scale. We then turned our attention to genome-level metabolic modelling (GSSM), which can predict the metabolic behaviour of organisms under different conditions, the production or consumption of metabolites, as well as the functions and effects of genes at the genome scale. So GSSM greatly broadens the scope of our modelling, and we can analyse the metabolic flow of DGF-298 as well as its polyploids on a genomic scale. We chose cobrapy as the GSSM analysis tool. In order to simulate metabolic flow on a larger scale, the commonly used tool is FBA, which assumes that the concentration of various metabolites is constant over time. And based on this, constraints are established and solved using linear programming.
Modular up-regulation of key genes based on metabolic network models:
Through simulations, we found that the effect of fold change in gene expression after polyploidisation on bacterial growth rate does not show a proportional relationship. We found that lower levels of two responses: NADH16 and PKF reduced the bacterial growth rate. We then selected the genes pfkB and nuoF, which encode these two responses, and constructed a plasmid containing these two genes and introduced DGF-298 to enhance the expression level of these two genes.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 690
Characterization：
We characterized these element combinations by measuring the expression intensity of the reporter gene gfp. To achieve this, we constructed four distinct plasmids: PACYAC-DM-GFP, PACYAC-DU-GFP, PACYAC-MU-GFP, and PACYAC-UD-GFP. Each plasmid was assembled by fusing the reporter gene gfp with various combinations of the regulatory elements J23109, J23110, J23116, and ribosome binding sites RBS33, RBS34, and RBS35. These constructs were seamlessly integrated into the PACYAC backbone using advanced DNA cloning techniques, allowing us to systematically explore the effects of these element combinations on gene expression. Since the nuoF and pfkB genes are located at different sites on the plasmid, it is not possible to use the gfp reporter gene for characterisation at the same time, so we integrated the gfp gene at different sites of the plasmid and carried out the characterisation several times successively. After characterising the expression intensity of different combinations of components, we inserted the nuoF and pfkB genes at specific sites, and the sequence of component combinations is shown below.
Protocal：
Our experimental conditions for characterizing this part were as follows:
- Plasmid Backbone: PACYC
- 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.)
Fluorescence intensity was detected using an enzyme marker at an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and fluorescence intensity values (a.u.) were obtained by calculating the fluorescence intensity to OD600 ratio.
Result
Access to synthetic biology components through the transcriptome：
Since the nuoF and pfkB genes are located at different sites on the plasmid and could not be characterised at the same time using the gfp reporter gene, we integrated the gfp gene at a different site on the plasmid and carried out two successive characterisations.
Changes in expression levels of different genes after modular regulation：
After transfection of nuoF and pfkB genes at different loci, RNA was extracted and subjected to reverse transcription PCR as well as qPCR for transcript level analysis
Strain growth after modular up-regulation of key genes：
We found that after modular up-regulation, only the combination of UD and MU led to an advancement of the logarithmic phase of growth in polyploid strains and partially restored the growth rate and biomass response, while other modes of regulation (e.g., DM and DU) did not show significant differences. We analyzed that the reason may lie in the existence of an extremely complex metabolic network in polyploid cells, where the nuoF gene and the pfkB gene catalyzed the electron transport and glycolysis, respectively. The regulation of pfkB gene is required to maintain a certain level of expression of nuoF gene in order to restore cell growth.
Reference：
[1] Ebrahim A, Lerman JA, Palsson BO, Hyduke DR. COBRApy: COnstraints-Based Reconstruction and Analysis for Python. BMC Syst Biol. 2013 Aug 8;7:74. doi: 10.1186/1752-0509-7-74. PMID: 23927696; PMCID: PMC3751080.
[2] Auriol C, Bestel-Corre G, Claude JB, Soucaille P, Meynial-Salles I. Stress-induced evolution of Escherichia coli points to original concepts in respiratory cofactor selectivity. Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1278-83. doi: 10.1073/pnas.1010431108. Epub 2011 Jan 4. PMID: 21205901; PMCID: PMC3029715.
[3] Lovingshimer MR, Siegele D, Reinhart GD. Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the expression and purification of phosphofructokinase from bacterial sources. Protein Expr Purif. 2006 Apr;46(2):475-82. doi: 10.1016/j.pep.2005.09.015. Epub 2005 Oct 18. PMID: 16289704.
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