Design

What is FarFarmIA?

This year, NTHU_Taiwan would like to address the overfertilization problem in global agriculture. Based on our design, our team genetically modified E. Coli so that it can produce different amounts of nutrients depending on the temperature changes in the environment. Therefore, we can efficiently nourish plants. In other words, giving the right amount of fertilizer to plants.

How does FarFarmIA bacteria work? It is equipped with thermal-sensitive lipase and adjustable fatty acid sensitive promoters. By combining different kinds of lipase (with different optimal responding temperature) and promoter, we can dynamically regulate the yield of downstream fertilizers. By doing so, Farfarmia bacteria can sense the temperature variation in the surrounding and secrete fertilizers correspondingly and precisely. Referring to the downstream, our engineered E.coli can turn urea into ammonium. Therefore, our “E.coli fertilizer “ is pure, ecofriendly, and even waste-recycling.

In short, We build three compartments into the sequence of E.coli: LipA, FadR regulator, and ammonium synthesis( through urease) system.

Figure 1. whole process

Compartment

A. Temperature-sensing biobrick---- cold-adapted lipase

Be able to catalyze the hydrolysis of triacylglycerol.
Having different activity at different temperatures.
Lipase of different bacteria and strains have varying reacting temperature zones.

Figure 2. the effect of ipaseA depends on temperature change

Overview of thermal-sensitive lipase

Lipases are glycerol ester hydrolases that are characterized by their ability to perform hydrolysis and turn triacylglycerols to free fatty acids and glycerol. Moreover, they are currently attracting enormous attention because of their biotechnological potential. Lipases occur widely in nature and are largely produced by animals, plants, and microbes; microbial lipases are the most studied and commercialized.

Microbial lipases have gained huge attention especially from industries because of their flexibility in temperature, pH, and organic solvent [1] . The characteristic raises our interest because it completely meets our needs of constructing a “bio-thermometer”. Below, we show a few lipases from different microbes as examples and their enzyme activities corresponding to reacting temperature zone respectively. As we mentioned above, TTSS can be customized to fit any temperature range at almost any place and dynamically regulate the yield of the downstream coding gene.

Antarctic Pseudomonas sp strain AMS3 [2] Exhibited broad temperature profile from 10–70 °C.
Pseudomonas sp. Lp1 [3] The observed pH and temperature range optimum for maximum lipase production were 8.0-8.5 and 37-42 °C, respectively.
Acinetobacter calcoaceticus 1-7 [4] The lipase exhibited optimum lipolytic activity at 40°C and has substantial activity from 20°C to 50°C.
Bacillus sp. [5] The enzyme showed maximum activity at pH 9 and temperature 60 °C.
Candida Antarctica [6] Candida antarctica produces two different lipases, named A (CALA) and B(CALB). CALA is an extremely thermostable protein, and the enzyme has its temperature optimum above 90 °C.

Figure 3. Few lipase from different microbes with various temperature-sensing range

In our project: lipase from Pseudomonas sp.7323

LipaseA from Antarctic cold-tolerant Pseudomonas sp. 7323 has a GXSXG motif, which is conserved in lipases/esterases and generally contains the active-site serine.

Notice that the catalytic activity of lipaseA changes at different temperatures because of conformational changes. Different from general lipase, cold-adapted lipase has the working temperature range that meets our need more appropriately. According to our reference [7], the low-temperature lipase gene can indeed be expressed in E. coli and the recombinant enzyme would have quite similar physicochemical properties with the wild-type lipaseA.

To purify the protein more easily, we expressed lipaseA protein with a 6× His-tagged fusion protein. Our E. coli also contains OmpA under the control of the lac Promoter(R0010), thus lipaseA can be expressed extracellularly by OmpA secretion signals at the N-terminal regions theoretically.

The graph below, based on the experimental data of lipase activity measurement in our reference, shows that the activity changes as temperature changes. The optimal temperature for the lipases activity is about 30°C.

In conclusion, the different temperature would trigger the change of fatty acid concentration based on the change of catalytic activity. Subsequently, different concentration of fatty acid can regulate the downstream gene-expression of fadBA.

Figure 4. the catalysis activity of lipase from Pseudomonas sp.7323 at different temperature

B. Fatty acid sensitive promoter

Overview of fadBA promoter

How can we undertake and respond to the fatty acid-producing by upstream lipaseA? Herein, we have designed a module to regulate the transcription of the fertilizer-producing enzyme with a fatty acid inducible promoter, FadBA [8].

In Escherichia coli, all the fatty acid is uptaken, activated and oxidized by the fatty acid degradation related gene such as fadA and fadB. The transcription of these related genes is well controlled by the FadBA promoter. This promoter is considered as a long-chain fatty acid (LCFA) sensor. It is repressed in the presence of fadR, a transcriptional regulator that can bind on the DNA and hence blocked the RNA polymerase to prevent the transcription of the gene. However, when the LCFA is transported through the membrane by fadL and then activated by fadD into acyl-coA, it can bind with the fadR, causing this repressor protein release from the promoters and transcription will be allowed.

Figure 5. fadBA promoter

fadBA promoter-editing

Actually, other iGEM teams such as NTU_Taida 2012 encountered problems with this promoter.

Massive leakage
The weakness

Since pFadBA has a massive leakage, the baseline expression of the reporter protein coupled with the promoter showed no obvious difference when induced with LCFA-coA. This makes the promoter becomes a poor sensor of fatty acid. NTU_Taida 2012 teams solved this problem with the overexpression fadR protein. Nevertheless, fadR overexpression is toxic to the cell since the normal fatty acid metabolism pathway will interfere. On the other hand, the strength of the promoter is weak.

At first, we thought that the native characteristic of this pfadBA promoter doesn’t quite meet our specifications to make a tunable promoter for our project, so we set out to improve it. But after seeing the modeled results we have other opinions about the various promoters we tested and designed. To ameliorate the weakness and leakiness of pfadBA, here is our modification on the promoter:

Figure 6. Ideal curvature of our promoter

Figure 7. overview Illustration of our modification

Edit the pribnow box with consensus sequence:

A consensus sequence is an ideal promoter sequence that can be found in the -10 and -35 sequence in the promoter region. We modify the pfadBA promoter with point mutation on the -10 and -35 box to give the greater and stronger expression of the downstream gene.
Add more repressor binding sites:

Herein, we expect to achieve the lower leakage.
1. fadBA promoter: We randomly insert more fadR binding sites and lac binding site into the promoter to help locking the promoter more tightly.
2. fadD promoter [9]: fadD promoter is naturally regulated by two fadR binding site( the sequence is slightly different from fadR binding site of fadBA promoter) and other repressors such as the oxygensensitive ArcA-ArcB two-component system and the cyclic AMP. We choose this promoter because we believe more repressor binding site could help locking the promoter more tightly and reducing the leakage. Except the wild type fadD promoter, we also replace the CRP repressor by fadR binding site or lac repressor.
Replace the pfadBA promoter with other native promoters exist in E. coli, and just reserve the original fadR binding site:

Herein, we expect to achieve the lower leakage and higher expression. Therefore, we use the wild type rrnD promoter with a UP element, which not only have greater strength but have no leakage. Later, we modify the rrnD promoter with fadR binding site of fadBA promoter.
Add the fatty acid producing gene (TesA ) before the fadBA promoter:

Herein, we expect to achieve the higher expression. Therefore, we use the wild type rrnD promoter with a UP element, which not only have greater strength but have no leakage. Later, we modify the rrnD promoter with fadR binding site of fadBA promoter.

The accumulation of Acyl-ACP, the metabolism product of carbon source such as fatty acid and glucose, in the cell will form negative feedback to the fatty acid biosynthesis. Thus, the TesA gene which encodes a thioesterase will hydrolyze these Acyl-ACP and produce a significant level of free fatty acid. Herein, we combine Tes A with pFadBA (wild type) together as a new promoter. By doing so, our promoter can be more sensitive to the change of fatty acid concentration.

For more information, please refer to the Partsin our wiki.

In conclusion, along with the catalytic activity changes of lipaseA at different temperatures, we can utilize the characteristic of this promoter to establish a dynamic expression system controlled by temperature since the different concentration of LCFA can induce different extent of gene expression.

C. Nutrients Assimilation

In this part, we develop a pathway which can convert human wastes into high-efficient nutrients, such as nitrogen fertilizer.

First, urease can catalyze the hydrolysis of urea into ammonia, then ammonia undergoes nitrification to form nitrite ions, which is a common nitrogen source for every plants and crop. Different from traditional and chemical fertilizer, these simple nutrients are more suitable for “smart fertilizer”.

References

LATIP, Wahhida, et al. Expression and characterization of thermotolerant lipase with broad pH profiles isolated from an Antarctic Pseudomonas sp strain AMS3. PeerJ, 2016, 4: e2420.
LATIP, Wahhida, et al. Expression and characterization of thermotolerant lipase with broad pH profiles isolated from an Antarctic Pseudomonas sp strain AMS3. PeerJ, 2016, 4: e2420.
KANIMOZHI, S.; PERINBAM, K. Optimization of Media Components and Growth Conditions to Enhance Lipase Production by Pseudomonas sp. Lp1. Biomedical and Pharmacology Journal, 2015, 3.2: 329-338.
WANG, Haikuan, et al. Screening and characterization of a novel alkaline lipase from Acinetobacter calcoaceticus 1-7 isolated from Bohai Bay in China for detergent formulation. Brazilian journal of microbiology, 2012, 43.1: 148-156.
GHORI, M. I.; IQBAL, M. J.; HAMEED, A. Characterization of a novel lipase from Bacillus sp. isolated from tannery wastes. Brazilian Journal of Microbiology, 2011, 42.1: 22-29
KIRK, Ole; CHRISTENSEN, Morten Würtz. Lipases from candida a ntarctica: unique biocatalysts from a unique origin. Organic Process Research & Development, 2002, 6.4: 446-451.
ZHANG, Jin-wei; ZENG, Run-ying. Molecular cloning and expression of a cold-adapted lipase gene from an Antarctic deep sea psychrotrophic bacterium Pseudomonas sp. 7323. Marine biotechnology, 2008, 10.5: 612-621.
https://2012.igem.org/Team:NTU-Taida/Result/pFadBA
FENG, Youjun; CRONAN, John E. Crosstalk of Escherichia coli FadR with global regulators in expression of fatty acid transport genes. PloS one, 2012, 7.9: e46275.
ZHANG, Fuzhong; CAROTHERS, James M.; KEASLING, Jay D. Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nature biotechnology, 2012, 30.4: 354.
PRESNELL, Kristin V.; FLEXER-HARRISON, Madeleine; ALPER, Hal S. Design and synthesis of synthetic UP elements for modulation of gene expression in Escherichia coli. Synthetic and systems biotechnology, 2019, 4.2: 99-106.
JENSEN, Peter Ruhdal; HAMMER, Karin. The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl. Environ. Microbiol., 1998, 64.1: 82-87.

CONTACT US

nthuxigem@gmail.com

Team:NTHU Taiwan/Design