Wet Lab Experiments
Welcome to the Wet Lab section of the HKUST(GZ) iGEM 2025 Wiki!
Wax Group:
The goal of waxset is to synthesize wax on the surface of litchi by Bacillus subtilis to enhance protection. We envisage two different stages of use: we cultivate the bacteria in the medium containing lactose, and spray the bacteria on the surface of litchi without lactose to synthesize wax and keep fresh. We used the expression characteristics of lactose operon to express fadr gene in the presence of lactose (when the bacteria were not inoculated on litchi) to promote fatty acid synthesis. At the same time, λ repressor inhibits the synthesis of a series of wax synthase (taking fatty acid as substrate), so as to achieve a large amount of fatty acid accumulation. In the absence of lactose (when bacteria were inoculated on the surface of litchi), the expression of the above genes was inhibited. The inhibitory effect of λ repressor disappeared, and the wax synthesis process downstream of fatty acids proceeded smoothly.
(the characteristic of lactose operon is that when there is no lactose, lacI will combine with LacO to inhibit the expression of downstream genes; When lactose is present, lactose will bind to lacI, so lacI cannot bind to LacO, and downstream genes can be normally expressed.)
Melatonin Group:
Our literature review indicates that melatonin has applications in fruit preservation due to its significant antioxidant properties. Furthermore, it can protect intracellular structures and mitigate damage caused by the attack of reactive oxygen species (ROS).
Based on these findings, we aim to genetically engineer bacteria to produce melatonin by expressing the series of enzymes involved in its synthesis pathway. We then plan to inoculate these engineered bacteria onto the surface of lychees. The intention is for the synthesized melatonin to be released into the surrounding environment via passive diffusion, thereby exerting a localized antioxidant effect.
The detailed synthesis steps are as follows.
Step 1: Tryptophan → 5-Hydroxytryptophan (5-HTP)
Enzyme involved: Tryptophan hydroxylase (TPH).
Significance: This is the initial step in melatonin synthesis. TPH catalyzes the addition of a hydroxyl group (-OH) to the 5th carbon of tryptophan's indole ring, forming 5-hydroxytryptophan. This product is the direct precursor for the subsequent synthesis of serotonin.
Step 2: 5-Hydroxytryptophan (5-HTP) → Serotonin (5-hydroxytryptamine, 5-HT)
Enzyme involved: Aromatic L-amino acid decarboxylase (AADC, also referred to as DDC).
Significance: The carboxyl group (-COOH) is removed from 5-hydroxytryptophan in a process called decarboxylation, yielding serotonin.
Step 3: Serotonin → N-acetylserotonin
Enzyme involved: Serotonin N-acetyltransferase (SNAT), more commonly known as Arylalkylamine N-acetyltransferase (AANAT).
Significance: This is the key regulatory step in melatonin synthesis. AANAT catalyzes the acetylation of serotonin by transferring an acetyl group from acetyl-CoA to serotonin's amino group (-NH₂), forming N-acetylserotonin. The activity of this enzyme is governed by the circadian rhythm, with significantly higher activity at night, which promotes melatonin synthesis.
Step 4: N-acetylserotonin → Melatonin
Enzyme involved: Acetylserotonin O-methyltransferase (ASMT), also known as Hydroxyindole-O-methyltransferase (HIOMT).
Significance: ASMT catalyzes the methylation of the 5-hydroxyl group (-OH) of N-acetylserotonin by adding a methyl group (-CH₃), ultimately forming melatonin.
Overview
This section documents all our laboratory work, including experiments, protocols, and results.
Experimental Design
[Describe the design of your experiments here]
Methods
[List and explain the methods used in your wet lab work]