Overview

Fig. 1. Overall design of Se coli
[click the picture for animation]

    With our goal to apply synthetic biology in space, Se coli, an engineered E. coli was designed to produce selenomelanin between its cell wall and cell membrane as protection from space radiation. Nevertheless, bacteria being able to resist space radiation cannot do us any good on its own. Taking that into consideration, NCKU_Tainan has decided to engineer our first version of Se coli to produce GABA, a substance capable of enhancing people’s sleeping quality and relieving melancholy.

    To synthesize selenomelanin, selenocysteine (Sec) and melanin need to be produced beforehand as the precursors. Since the commercial Sec is expensive and the resources in space are limited, a stable, scalable, and sustainable method must be found to produce Sec. In vitro and in vivo strategies through the pathways of tRNA and cysteine (Cys) were tried respectively. On the other hand, melA was overexpressed to encode for tyrosinase which catalyzes the oxidation from tyrosine (Tyr) to melanin. Sec and melanin were then put together to form selenomelanin. Moreover, the function test (see Measurement page) examines the function of selenomelanin to prove its ability to protect microorganisms.

    To further test the radiation resistance of Se coli, MerStage was designed as the hardware to mimic cell performances in space and examine the function of Se coli under UV light (see Hardware page).

    For biosafety concerns, tyrA was knocked out, so that Se coli cannot produce Tyr. This way, it can only live in medium or plates with additional Tyr, preventing it from spreading to the environment.

    Integrating all the design and engineering processes mentioned above, we are determined to provide and establish a systematic way for selenomelanin synthesis, to make space exploration easier.

Selenocysteine Synthesis

In Vitro Strategy: tRNA Pathway

Fig. 2. tRNA pathway for Sec synthesis
[click the picture for animation]

    The in vitro strategy involves the utilization of tRNAs to synthesize Sec from serine (Ser) with certain enzymes and the deacylation of selenocysteinyl-tRNA to obtain a single Sec molecule for higher purification.

    To begin with, three different tRNAs were selected: tRNA^Sec (BBa_K4171008), the original tRNA in the natural pathway[1]; tRNA^UTuX (BBa_K4171009), the modified strain of tRNA^Sec which has a higher affinity to SelA (BBa_K4171002), the L-seryl-tRNA^Sec selenium transferase; and tRNA^Ser (BBa_K4171007), a natural tRNA with a high affinity to Ser that can attach and incorporate it into proteins[2]. Ser was then attached to these three kinds of tRNAs in the presence of the seryl-tRNA synthetase SerRS (BBa_K4171001)[3, 4]. Samples were extracted several times to get more purified seryl-tRNAs, which could be further converted into selenocysteinyl-tRNA with 6 additional substances: sodium selenite, SelA[5], selenophosphate synthetase (SelD) (BBa_K4171003)[6], ATP, DTT, and seryl-tRNAs. Samples were dried in vacuo after purification[7].

    Deacylation of selenocysteinyl-tRNAs then took place, with either Tris-HCl or nuclease S1, and 10 kDa columns were used for filtration[8, 9]. After being deacylated, samples were analyzed using High-Performance Liquid Chromatography (HPLC).

In Vivo Strategy: Cysteine Synthesis Pathway

    To simplify the number of components required in the in vitro strategy, in vivo strategy was developed by synthesizing Sec through the Cys synthesis pathway. Wild-type E. coli is capable of producing Cys with the presence of Ser and sulfide sources like sulfate, sulfite, and sulfide[10]. Furthermore, specific enzymes that originally produce Cys can synthesize Sec by replacing sulfide with selenide[11]. Sodium selenite was therefore added in this pathway to intrude the sulfate metabolism pathway, and then Sec is expected to be produced instead of Cys.

Fig. 3. In vivo pathway for Sec synthesis
[click the picture for animation]

I. cysK and cysE* genes

    cysK and cysE* were overexpressed to produce Sec from Ser and sodium selenite. cysE, the gene encoding for serine acetyltransferase, transforms Ser into one of the precursors of Sec called O-acetyl-L-serine (OAS). However, in the Cys synthesis pathway, CysE is inhibited by excessive Cys. CysE might still be downregulated by the synthesis of Sec, although sulfide has been replaced by selenide. To increase the total production of Sec and avoid negative feedback from Cys and Sec, some mutations were introduced, forming CysE* (BBa_K4171011)[12]. CysK (BBa_K4171010), the Cys synthase, turns OAS into Cys when hydrogen sulfide is present. Therefore, when given sodium selenite instead of hydrogen sulfide, CysK was expected to synthesize Sec.

II. YdeD membrane protein

    There are two major difficulties in Sec production: Sec accumulation and extraction. The accumulation of excessive Sec decreases cell growth, and the total production of Sec will then drop. Aside from that, it is necessary to break cell membrane through sonication to extract Sec, but it is difficult to separate Sec from other amino acids and the disrupted cell.

    To solve these problems, YdeD was added into this pathway. YdeD (BBa_K4171005), the transmembrane protein, serves as an export pump for Cys and other metabolites of the Cys synthesis pathway[13]. With YdeD, E. coli automatically pumps out Sec, making the cell a natural factory of Sec synthesis.

III. IscS

    The cysteine desulfurase IscS degrades Cys, as well as Sec, and then converts it into alanine[14]. To maximize Sec production, iscS was knocked out.

IV. Sec preparation and analysis

    After the function of all the proteins was measured and the best strain was chosen, experiments were constructed with sodium selenite through the carefully designed protocol. The samples were analyzed via HPLC.

Melanin and Selenomelanin Synthesis

Fig. 4. Melanin synthesis pathway
[click the picture for animation]

    Aside from Sec, melanin is another precursor to produce selenomelanin. Since Sec is harder to synthesize, the synthesis of melanin was first conducted and optimized, then followed by Sec addition to produce selenomelanin.

     By overexpressing TyrP and MelA (BBa_K4171024), we successfully increased the total production of melanin through importing more Tyr (see Improvement page). TyrP serves as the Tyr transporter and MelA is the tyrosinase that catalyzes Tyr into melanin. As Sec being added to the melanin-producing E. coli, they together polymerize into selenomelanin.

Melanin and Selenomelanin Function Test

Fig. 5. Function test design for melanin and selenomelanin
[click the picture for animation]

    Melanin has been proven to protect cells from oxidative stress caused by radiation, such as UV light. Therefore, UV experiments were conducted to examine melanin function, by comparing the survival rate of melanized bacteria and that of non-melanized bacteria under UV radiation exposure to confirm its radioactive protection.

    First, the bacteria (melanized and non-melanized) on the plate were exposed to the UV light (UV-B, 24 W) for 5 minutes. To control the total amount of the cells, OD₆₀₀ value was adjusted by dilution after the bacteria were washed down from the plate with LB medium. Samples were then diluted into different dilution ratios, while 5 μL of each ratio was applied onto agar plates. After 37℃ overnight incubation, the colony forming unit (CFU) was calculated to compare the survival rate of melanized and non-melanized bacteria.

    The effect of Se coli was verified through the same measuring methods. The radiation resistance of Se coli was examined by comparing its survival rate with that of melanized and non-melanized cells, and it was expected to have the best survivability under radiation among the three groups. By testing the radiation resistance of Se coli, the presence of selenomelanin was also demonstrated.

GABA Synthesis

Fig. 6. GABA synthesis pathway
[click the picture for animation]

    To demonstrate the usage of Se coli, MelA and GAD were overexpressed to let Se coli produce selenomelanin and GABA in vivo.

    GABA synthesis pathway involves monosodium glutamate (MSG) as precursor and glutamate decarboxylase (GAD) from gadB gene as catalyst. Plasmid containing gadB (BBa_K4171023) was transformed into Se coli. The result not only indicated that the engineered bacteria had a higher UV resistance but also showed its helpfulness in the production of useful chemical substances.

Biosafety

Fig. 7. The design of biosafety

    Since we aim to engineer E. coli with resistance to radiation, there is a risk of bacteria leaking out from laminar bench cabinets even with UV-C sterilization. To control the risk of bacteria leakage, tyrA was knocked out to block the formation of Tyr. Since Tyr is indispensable for not only melanin synthesis but also bacteria survival, tyrA deletion will let E. coli survive only when Tyr is added (see Safety page).

Device

Fig. 8. MerStage

Fig. 9. Hanging drop microfluidic chip

    Before sending Se coli to space, its radiation resistance and maximum functionality must be confirmed. To test the function of Se coli, a stage device was designed consisting of two parts: the radiation source and the hanging drop microfluidic chip. In addition to the radiation source, a hanging drop microfluidic chip was designed to promote bacteria aggregation and 3D structure formation, which are the typical cell performances in space[15].

    After testing different well sizes of the microfluidic chip and observing the bacteria aggregation under the microscope, the microfluidic chip and the radiation sources were combined to build MerStage, and we used it to test bacteria radiation resistance with typical performances in space.