DBTL(Design-Build-Test-Learn) cycle We adopted the DBTL cycle and tried to make it reproducible by others by showing the basic and composite parts used and the lab notebook.

1-1.Design

We designed a pathway to artificially produce methane in three steps: to breakdown of cellulose in cattle manure to glucose, to generate of acetic acid from glucose, and to produce of methane from it. We focused on creating acetic acid that is needed as a reaction substance for methanogens of these three processes.

1-2. Build

Next, we designed the parts that hydrolyze cellulose to glucose. We introduced the genes of four enzymes, BBa_K3394000:Endo5a cellulase , BBa_K1499501:Endo-1,4-beta-glucanase, BBa_K2449012:exo-1, 4-glucanase , BBa_K1131002:B-Grucpsodase, which is needed for hydrolysis into pH1 T7 Flexi® Vectors and transformed E. coli. Then we added cellulose samples to the culture medium, and verified that glucose could be detected by TLC. We added cow manure containing cellulose to the culture medium as well.

1-3. Test

Although we have not actually experimented with it, an expert who represents a thermostable enzyme laboratory company which is involved in synthetic biology in Japan told us to create a more efficient cellulase preparation instead of using the plasmid. In addition, he advised us on the direction of this project.

1-4. Learn

We learned that the demand for cellulase mass production was not so great, whereas an efficient synthesis of acetic acid has not been established yet. Therefore, we decided to focus on how the acetic acid worked as one of the three processes.

2-1Design

We decided to create an E. coli which produces acetic acid. In the process, we introduced a gene, PoxB, which would make more acetic acid in vivo and to attach another gene, AatA, as a pump to the E. coli to discharge acetic acid.

2-2Build

Acetic acid was presumed to be toxic to E. coli. According to Stéphane Pinhal et al. (2019), high concentrations of acetate inhibit growth of E. coli because it causes the perturbation of acetate metabolism. Therefore, we decided to create the modeling.

2-3Test

Next, we predicted that the E. coli died depending on the amount of gene expression from the results of modeling.

2-4Learn

Since it was suggested that IPTG-induced preparation of Pox B expression might be necessary, we eventually thought it would be better to create a vector with an ever-expressing promoter and created a plasmid that enabled PoxB to be adjusted with design IPTG.

3-1. design

We created an E. Coli producing acetate. PoxB is regulated by IPTG and aatA always expresses. To find the optimal promoter strength, adjust the IPTG concentration and measure the amount of acetic acid produced and the growth rate of E. coli.

3-2. build

Incorporate poxB and aatA into the expression plasmid, pTWV228, and express in E.coli. Pox B is placed downstream of the lac promoter. AatA is placed downstream of the constitutive promoter.

3-3. test

We cultured E. Coli at different iptg concentrations and determined OD 600 and acetic acid concentrations. OD 600 increased rapidly at higher IPTG concentrations(graph.1), and acetate was detected by acetate assay kit in the samples with IPTG 1,5mM(graph.2).

3-4. learn

Millard et al. (2022) have argued that acetate acts as a co-substrate of glycolytic nutrients and boosts E. coli growth at low glycolytic fluxes. The change in OD600 with the addition of IPTG may be due to increased intracellular acetate concentration and growth promotion by the expression of poxB. we have confirmed the production of acetic acid, we need to use these data to explore the optimal promoter strength of poxB.

References

Millard, P., Uttenweiler-Joseph, S., & Enjalbert, B. (2022). From toxic waste to beneficial nutrient: acetate boosts Escherichia coli growth at low glycolytic flux. BioRxiv. https://doi.org/10.1101/2022.09.20.506926