Our team harnessed the power of EPS, extracellular polysaccharides, to enhance viscosity.
Through experiments, we discovered their potential to mitigate soil desertification by improving soil adhesion. For
example, future teams could introduce EPS into arid and cracked soil to increase the adhesion between soil particles,
making it suitable for plant growth and ultimately combating soil desertification. Unexpectedly, during our experiments,
we found that EPS not only increased viscosity but also exhibited cholesterol adsorption capabilities. Consequently, it
can play a role in cholesterol degradation. For future teams working on cholesterol degradation, direct utilization of
EPS could be considered.

In our experiments, we employed a low-temperature inducible promoter to enable efficient gene
expression under low-temperature conditions. Often, environmental temperatures may not match the ideal lab temperature
(37 degrees Celsius), making the low-temperature inducible promoter a valuable tool to conduct projects under a wider
range of environmental conditions. For future teams encountering suboptimal temperatures for enzyme reactions, the
integration of a low-temperature inducible promoter upstream of target genes in plasmids can enhance gene expression
levels.

In the MICP process, microorganisms such as Sporosarcina pasteurii hydrolyze urea by
expressing urease, thereby rapidly increasing the pH value and the concentration of carbonate in the cell
microenvironment, forming an alkaline environment necessary for inducing calcium carbonate precipitation.

The surface of microbial cells usually has a large number of negatively charged functional
groups, which adsorb the positively charged Ca2+ in the solution. Ca2+ will form calcium carbonate when it meets high
concentration of carbonate and precipitates on the cell surface. With the expansion of calcium carbonate precipitation,
the microorganisms will be gradually wrapped, limiting the diffusion of their nutrients, resulting in microbial death
and eventually forming biommineralized bodies. This is the whole chemical reaction process.

Reasons for selection: The number of urease genes is relatively small, and only ureA, ureB and ureC are composed of
three structural genes, which can be assembled into a complete urease to play a role without other auxiliary genes and
regulatory genes, that is, there are fewer interference items.

The above diagram shows the composition of urease genes from different sources


For the future team, urease can decompose urea to produce ammonia gas and provide raw materials for the production of
nitrogen fertilizer. Similarly, urease can be applied in the treatment of sewage. If the project involves sewage
treatment, urease can be directly used to decompose nitrogen-containing organic matter in sewage to prevent
eutrophication in the discharged waters.

Carbonic anhydrase (CA) is a class of zinc-containing enzymes capable of efficiently
catalyzing the reversible reaction between carbon dioxide, water, bicarbonate, and H+ ions. CA maintains high activity
and stability under conditions with a pH range of 4.0–9.0 and temperatures below 65°C. The catalytic rate of carbonic
anhydrase is exceptionally rapid, typically ranging from (10^4 –10^6 )/s across various families. Therefore, it holds
promise for significantly enhancing the rate of biomineralization in Microbially Induced Calcium Carbonate Precipitation
(MICP), garnering considerable attention.
As a biological catalyst, future teams seeking high rates of calcium carbonate precipitation can directly employ
carbonic anhydrase to accelerate processes such as biocement drying, enhancing user experiences, and more.

Under natural circumstances, the rate of CO2 hydration is exceedingly low, approximately
1.3×10–1/s, which greatly limits the production of calcium carbonate precipitation, the primary reason for the slow pace
of MICP reactions. In contrast, natural carbonic anhydrase achieves the highest CO2 hydration rate, reaching up to
approximately 1.4×10^7 /s, representing an enhancement of around 10^8 times compared to natural conditions.
Consequently, the catalytic action of carbonic anhydrase effectively accelerates biomineralization rates. The CO2
hydration process involves the following stages:

In conclusion, our contributions encompass the application of EPS for soil improvement and
cholesterol degradation, the utilization of a low-temperature inducible promoter for versatile gene expression, and the
exploration of enzymes like urease and carbonic anhydrase for MICP and other biomineralization processes. Our work
serves as a foundational platform from which future iGEM teams can innovate and address environmental and
biotechnological challenges. By sharing our findings, we aim to empower and inspire future teams to continue advancing
the field of synthetic biology for the benefit of the environment and society.