Overview
When developing the project, NCKU_Tainan has tried some new experiment designs and tools to optimize the production to inspire and benefit other iGEM teams. In Sec synthesis, a system to synthesize Sec through the in vitro tRNA pathway was established by simplifying the protocols collected from various papers. In addition, we also dedicated ourselves to realizing in vivo Sec synthesis. We discovered that Sec and Cys could be easily distinguished by HPLC, while others usually use LC-MS, a much more complex, demanding and expensive way. Even though yielding Sec through in vivo pathway failed, the function of YdeD was confirmed. In selenomelanin synthesis, Taguchi methods were introduced when producing melanin and survival rate tests were utilized to see the ability of selenomelanin and melanin protection. Last, MerStage was designed as the measuring platform. Through these efforts, NCKU_Tainan hopes that the contributions can assist future teams when they have similar demands or meet similar difficulties; furthermore, solving more problems in our world.
Selenocysteine Synthesis
Sec is the 21st amino acid and is extremely expensive in commercial products. In the project, NCKU_Tainan designed two pathways to synthesize this valuable amino acid and analyze it through HPLC.
In Vitro Selenocysteine Synthesis
NCKU_Tainan provided another Sec synthesis pathway, which was conducted in vitro. The experimental conditions were simplified since the absence of interference from other molecules in the intact cell. The process would therefore be easier to control, and the production of Sec would be purer. The in vitro Sec synthesis pathway can be divided into three processes: First, attaching Ser to three species of tRNAs; second, converting seryl-tRNA to selenocysteinyl-tRNA by the enzymes SelD and SelA; third, deacylating Sec from the tRNAs. After these three steps, Sec could be produced and measured using HPLC.
The methods adopted were mainly based on past research, but protocols and materials were adjusted. Hence, it will be easier for future iGEM teams to conduct the experiments by following the optimized and simplified protocols. For tRNA in vitro synthesis, the reannealing protocol was added to our method to ensure that the tRNAs were refolded correctly. In addition, two ways of deacylation to deacylate Sec from the tRNAs were introduced: adding Tris-HCl (pH 9.5) to break the ester bond between amino acid and tRNA under alkaline conditions and the addition of nuclease S1 to cut intact tRNAs into pieces (see Experiments page).
Through this pathway, Sec has been successfully produced. As the results show, by using tRNASer and tRNAUTuX NCKU_Tainan succeeded in producing Sec (Fig. 1).
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Fig. 1. Confirmation of Sec by HPLC (A) Sec synthesized by tRNASer; (B) Sec synthesized by tRNAUTuX.
HPLC Assay
NCKU_Tainan discovered that Sec and Cys could be easily distinguished by HPLC assay, for the peak of Cys showed up around 8 minutes while that of Sec appeared at about 10 minutes. It is difficult for students to apply LC-MS for measurements since it sets many requirements for the sample. Herein, HPLC seems to be much more suitable for analysis.
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Fig. 2. HPLC result of standard Cys Fig. 3. HPLC result of standard Sec.
In vivo Selenocysteine Synthesis
YdeD (BBa_K4171005)
Though Sec synthesis through in vivo pathway failed, NCKU_Tainan successfully proved the function of YdeD. ydeD (BBa_K4171005) is the gene expressing YdeD, a membrane protein which is responsible for pumping out Cys and Sec as well as maintaining the metabolism of E. coli. It is noticed that with ydeD overexpressed, the survival rate of E. coli was higher than those without ydeD when fed with sodium selenite (Fig. 4).
Fig. 4. CFU results examining the function of YdeD
Furthermore, through HPLC analysis, YdeD was discovered to help E. coli utilize Ser more efficiently (Fig. 5), proving that YdeD is engaged in some metabolic pathways for Ser degradation or transformation.
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Fig. 5. Confirmation of result by HPLC (A) PlacI-cysK-cysE*/MG1655 cultured with Na2SO3; (B) PlacI-cysK-cysE*/MG1655 cultured with Na2SeO3; (C) PlacI-cysK-cysE*, PompA-ydeD/MG1655 cultured with Na2SO3; (D) PlacI-cysK-cysE*, PompA-ydeD/MG1655 cultured with Na2SeO3.
As a result, NCKU_Tainan believes that the discovery of a simplified Sec in vitro synthesis method, HPLC analysis for distinguishing Sec and other amino acids and the function of YdeD can contribute to future experiments and studies.
Melanin Synthesis
Taguchi Methods
To find the best reaction condition to produce melanin, such as different mediums or concentrations of the cofactors, NCKU_Tainan decided to use the Taguchi methods, a popular quality control approach in engineering and manufacturing. The basic idea of the Taguchi methods is to use statistical and mathematical methods to extract more information from fewer experiments. These methods are beneficial and efficient in minimizing the variation of quality characteristics and producing high-quality products.
By Taguchi methods, NCKU_Tainan has reached the goal of optimizing manufacturing parameters and comparing factor significance. For future iGEM teams that want to optimize the bioproduct synthesis, NCKU_Tainan has set up an example of applying Taguchi methods in their project to increase production yield (see Model page).
Survival Rate Test
To determine the ability of melanin or selenomelanin protection, NCKU_Tainan designed a method to calculate the survival rate of E. coli after being irradiated with UV light.
The experiment was divided into three processes: irradiation, dilution, and dropping.
First, the plates with bacteria colonies were exposed to UV light for 5 minutes, then the cells were washed down with LBYT medium. After adjusting the OD600 value to 1, the samples were diluted sequentially from 10 to 106 times.
Next, 5 μL of each sample was dropped onto the agar plates, and the plates were cultured at 37℃ overnight.
Finally, the number of colonies was counted, and the survival rate was calculated by dividing UV-irradiated CFUs by non UV-irradiated CFUs.
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Fig. 6. Bacteria under exposure of UV-B. (A) Survival rate (B) CFU comparison.
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Fig. 7. Bacteria under exposure of UV-C (A) Survival rate (B) CFU comparison.
The success of the above measuring method built an available measuring platform for future iGEM teams. Moreover, it indicates that bacteria with better UV tolerance had been created for broader use.
MerStage
Fig. 8. How we made MerStage
MerStage is our hardware device, it can simulate bacteria performance in a microgravity environment condition in a simpler and cheaper way. Since the microgravity environment is hard to create on the earth, MerStage can simulate E. coli under space environment such as bacteria aggregation and 3D culture.
MerStage consists of two components, one is a hanging drop microfluidic chip which is used to simulate the performance of the microorganism under space conditions, and the other one is UV-C sources, a kind of harmful short-wavelngth radiation that only exists in space, which are used to test if the bacteria can survive under irradiation.
If future iGEM teams engineer other radiation-proof microorganisms and would like to send them to space, MerStage can provide an innovative method for the teams to test the radiation resistance.
