Selenocysteine

In Vitro Pathway

I. tRNA in vitro synthesis

Achievements:

1. Preparation of the DNA template for in vitro tRNA synthesis
2. tRNA in vitro synthesis

Process:

    T7 RiboMAX^TM Express Large Scale RNA Production System (from Promega Corporation) was used to synthesize tRNA in vitro. Before synthesizing tRNA, DNA templates were prepared for the three tRNA species, which were tRNA^Ser, tRNA^Sec, and tRNA^UTuX.

    First, the tRNA sequences ordered from IDT were constructed, with T7 promoter in the beginning of the sequence, into TA vectors respectively.

    Next, after the constructed plasmid was successfully transformed into E. coli DH5α, we repeated the process: E. coli culture, plasmid extraction, plasmid digestion, and gene amplification by PCR.

    After PCR, all the products were condensed as the final template for tRNA in vitro synthesis, and the preparation of DNA templates was done (Fig. 1).

Fig. 1. Confirmation of P_T7 with tRNA^Ser (BBa_K4171014), tRNA^Sec (BBa_K4171015), and tRNA^UTuX (BBa_K4171016) sequence construction by PCR
M: Marker; Lane1: P_T7-tRNA^Ser (111 bp); Lane2: P_T7-tRNA^Sec (118 bp); Lane3: P_T7-tRNA^UTuX (114 bp)

    T7 RiboMAX^TM Express Large Scale RNA Production System was used to synthesize tRNA, which is the substrate of the next experiment (check out aminoacylation protocol in Experiments page) (Fig. 2).

Fig. 2. Confirmation of tRNA^Ser (BBa_K4171007), tRNA^Sec (BBa_K4171008), and tRNA^UTuX (BBa_K4171009) in vitro synthesis
M: Marker; Lane1: tRNA^Ser (88 bp); Lane2: tRNA^Sec (95 bp); Lane3: tRNA^UTuX (91 bp)

II. Selenocysteine synthesis

Achievements:

1. Aminoacylation
2. Conversion of seryl-tRNA to selenocysteinyl-tRNA
3. Deacylation

Process:

    To synthesize Sec, we imitated the native Sec synthesis pathway from E. coli. Three enzymes were needed in selenocysteinyl-tRNA production: SerRS, SelD, and SelA. SerRS (BBa_K4171001) was used in seryl-tRNA production, while SelA (BBa_K4171002) and SelD (BBa_K4171003) were used to convert seryl-tRNA into selenocysteinyl-tRNA[1, 2, 3].

    The protocol based on the accompanying paper[4] was followed (see Experiments page), and tRNA was made to bind with Ser through enzyme SerRS and buffer (see Experiments page). Urea PAGE was used to verify the size difference between tRNA and aminoacylated tRNA as a confirmation of aminoacylation (Fig. 3 and 4).

    After aminoacylation, seryl-tRNA was converted to selenocysteinyl-tRNA with enzymes SelA and SelD. The protocol was based on the accompanying paper[5] (see Experiments page), and the result of Urea PAGE is shown below (Fig. 3 and 4).

Fig. 3. Confirmation of the result after aminoacylation and conversion of seryl-tRNA to selenocysteinyl-tRNA by Urea PAGE
Lane1: tRNA^Ser; Lane2: seryl-tRNA^Ser; Lane3: selenocysteinyl-tRNA^Ser; Lane4: tRNA^Sec; Lane5: seryl-tRNA^Sec; Lane6: selenocysteinyl-tRNA^Sec

Fig. 4. Confirmation of the result after aminoacylation and conversion of seryl-tRNA to selenocysteinyl-tRNA by Urea PAGE
Lane1: tRNA^UTuX; Lane2: seryl-tRNA^UTuX; Lane3: selenocysteinyl-tRNA^UTuX

     The bands of seryl-tRNA and selenocysteinyl-tRNA have upshifted compared to the band of the tRNA. The result shows that the amino acid was successfully charged to the tRNA.

    For the final step, deacylation was conducted. Two methods were tried: Tris-HCl[6] and nuclease S1[7] (see Experiments page). After deacylation, HPLC was used to see the results (Fig. 5). As the results show, we had successfully synthesized Sec through the in vitro pathway. In our experiment results, the peaks of Sec made from tRNA^Ser and tRNA^UTuX are similar to those of Sec standard, which proves that tRNA in vitro Sec synthesis is successful.

                (A)

                (B)

Fig. 5. Confirmation of Sec by HPLC (A) Sec synthesized by tRNA^Ser; (B) Sec synthesized by tRNA^UTuX

    In these results, the retention time of the Sec standard is 10.5 minutes (the peaks at 3.5 minutes and 4.5 minutes are solvent peaks), which is the same as the retention time of Sec synthesized via the in vitro pathway.

Table 1. Comparison between the results of the three tRNA species in aminoacylation
and conversion of seryl-tRNA to selenocysteinyl-tRNA.

    Based on the result shown in Table 1, it is concluded that by using tRNA^Ser and tRNA^UTuX, in vitro Sec synthesis method can succeed. There are still further experiments required to prove whether it can succeed in using tRNA^Sec.

In Vivo Pathway

I. Construction success

Achievements:

1. Verification of CysK and CysE* function
2. Construction of pSU-P_lacI-cysK-cysE* (BBa_K4171012)
3. Construction of pP_ompA-ydeD (BBa_K4171013)

    To synthesize Sec in vivo, we planned to take advantage of the natural Cys synthesis pathway and substitute sulfide with selenide as one of the substrates. Thus, cysE* and cysK were overexpressed in order to yield Sec.

     To evaluate CysK and CysE* function, P_lacI-cysK and P_lacI-cysE* were transformed into E. coli strains with cysK and cysE being mutated (△cysK and △cysE) and the growth curves were measured. Theoretically, once we successfully transformed the plasmids into E. coli △cysK and △cysE mutant, their growth curves should become similar to that of wild-type’s. Results (Fig. 6 and Fig. 7) showed that the transformed plasmids compensated for the mutated genes, which proved CysK and CysE* functions.

Fig. 6. Examination of CysK function by transforming PlacI-cysK (BBa_K4171010) into E. coli △cysK mutant

Fig. 7. Examination of CysE* function by transforming PlacI-cysE* (BBa_K4171011) into E. coli △cysE mutant

    Then, we attempted to ligate cysK and cysE* on the same plasmid, and transformed it to E. coli MG1655. The structure was confirmed by colony PCR.

Fig. 8. Confirmation of P_lacI-cysK-cysE* by colony PCR
M: Marker; Lane 1: P_lacI-cysK-cysE* (2259bp)

    Furthermore, we constructed ydeD, the gene encodes for the transmembrane protein, with ompA promoter in another plasmid, and confirmed its structure with double digestion. By incorporating P_ompA-ydeD into the pathway, the total production of Sec is expected to increase, and E. coli should grow better.

Fig. 9. Confirmation of P_ompA-ydeD (BBa_K4171013) by colony PCR
M: Marker; Lane 1: P_ompA-ydeD (1267bp)

II. Optimization of engineered E. coli

Achievements:

1. Comparison between E. coli MG1655 and △iscS
2. Applying CFU to examine YdeD function

Process:

    After successful construction, pSU-P_lacI-cysK-cysE* and P_ompA-ydeD were transformed into wild-type E. coli MG1655 and △iscS. IscS engages in Cys degradation pathway, which is supposed to degrade Sec. Two kinds of engineered E. coli were cultured with sulfide, and their growth curves were measured. E. coli △iscS barely grew compared to MG1655 based on the result (Fig. 10), which might be caused by excessive Cys accumulating within the cells. Thus, we chose MG1655 for further experiments. Then, we started to culture E. coli with sodium selenite. The medium turned red since selenide was deoxidized to selenium by bacteria[8]. It was noticed that with ydeD overexpression, the color of the medium was lighter (Fig. 11).

Fig. 10. Strain selection by measuring growth curves

Fig. 11. Bacteria cultured in different concentrations of sodium selenite

    We, therefore, hypothesized that the production of Sec increased since YdeD pumped it out efficiently and the total amount of selenium decreased subsequently. Extending this hypothesis, E. coli should grow better with YdeD, for excessive Sec contains toxicity. To verify it, CFU was conducted to estimate the numbers of bacteria and to examine YdeD function. E. coli was cultured with both sodium selenite and sodium sulfite, while wild-type E. coli with and without ydeD was tested as the control group. Based on the result (Fig. 12), only the cell numbers of groups 7 and 8 showed an obvious difference, which proves two things. First, it was sodium selenite and its metabolites that contributed to the difference in the survival of E. coli. Second, the function of YdeD was not that obvious without CysK and CysE* in groups 3 and 4, since Sec was not synthesized as much as those in 7 and 8. Combining this information, we successfully evaluated YdeD function.

Fig. 12. CFU results examining the function of YdeD

III. Sec preparation and measurements

Achievements:

1. Sec preparation
2. Applying HPLC to examine the synthesis of Sec

Process:

    The protocol of how E. coli were cultured to yield Sec is as follows:
    Step 1. Culture E. coli in M9 with 10% LB
    Step 2. Add 5 mM sodium selenite 4 hours later than Step 1
    Step 3. Harvest the culture 24 hours later than Step 2


    We followed some papers to establish this protocol. To begin with, research has shown that once the concentration of selenide is over 0.08 mM, the growth of bacteria will be inhibited[8]. However, in an attempt to yield more Sec, 5 mM sodium selenite was still added and it took 4 hours to get enough amount of E. coli. Moreover, another research has shown that after around 20 hours of culturing, the production of Cys increased significantly[9], which is the reason contributing to the 24-hour culturing in the last step.

    The samples were then prepared for HPLC analysis to test whether Sec was produced. We also cultured E. coli without ydeD overexpression to test the ability of YdeD. All the strains were cultured in sodium sulfite as well as the control groups and only the supernatant was kept for analysis. Standard Cys (Fig. 13) and Sec (Fig. 5) and Ser (Fig. 13) were also sent to HPLC, and the time for their peaks were around 8, 10, and 5 minutes respectively.

    Unfortunately, this pathway seemed to have failed and Sec might not have been synthesized according to the results. E. coli that were cultured with sodium sulfite (Fig. 14 A and C) should produce Cys, but a clear peak around 8 mins was barely observed. Meanwhile, E. coli cultured with sodium selenite (Fig. 14 B and D) should produce Sec. Although there were peaks around 10 minutes, the peaks still existed even without sodium selenite, the substrate for Sec. We assumed that it was because the probability for selenide to intrude sulfide metabolism pathway was much lower than our expectation, so that E. coli did not efficiently convert sodium selenite into Sec. Another possibility was that sodium selenite had been mostly reduced to selenium before being incorporated into the cysteine synthesis pathway, which turned the medium red.

    There was another thing that drew our attention. Comparing the results with ydeD expression (Fig. 14 C and D) and those without (Fig. 14 A and B), Ser decreased significantly when E. coli translated YdeD at large. YdeD may have made E. coli degrade Ser more efficiently, but the results and the mechanisms behind are not clear. In the future, we would like to modify this pathway, either searching for more papers for adjustments or trying to tackle the two problems mentioned above.

(A)

(B)

Fig. 13. HPLC results of standard Cys and Ser (A) Standard Cys; (B) Standard Ser

(A)	(B)
(C)	(D)

Fig. 14. Confirmation of results by HPLC (A) P_lacI-cysK-cysE*/MG1655 cultured with Na₂SO₃; (B) P_lacI-cysK-cysE*/MG1655 cultured with Na₂SeO₃; (C) P_lacI-cysK-cysE*, P_ompA-ydeD/MG1655 cultured with Na₂SO₃; (D) P_lacI-cysK-cysE*, P_ompA-ydeD/MG1655 cultured with Na₂SeO₃

Melanin

Tyrosinase Synthesis

Achievement:

1. Construction of plasmids containing melA genes

Process:

    To synthesize melanin, tyrosinase is the critical enzyme catalyzing the reaction. Hence, we cloned melA (BBa_K274001) into pSB4KI backbone driven by Trc promoter for melanin production. After transforming into commercial E. coli DH5α competent cell, we conducted confirmation to verify whether E. coli uptook the plasmid. The size of the plasmid was correct, indicating the construction was successful. In addition, we tested the protein expression of pSB4KI-P_trc-melA in DH5α by SDS-PAGE, and the result is shown below.

Fig. 15. Confirmation of pSB4KI-P_trc-melA (BBa_K4171018) by digestion
M: Marker; Lane 1: pSB4KI-P_trc-melA (6707 bp); Lane 2: pSB4KI-P_trc-melA without digestion (negative control)

    As Fig. 16 shows, there was slightly more MelA protein expression compared with wild-type DH5α, demonstrating that we had successfully expressed exogenous melA gene in E. coli.

Fig. 16. The SDS-PAGE result of pSB4KI-P_trc-melA (BBa_K4171018) in MelA (67 kDa) expression with and without IPTG induction
M: Marker; Lane 1: With IPTG induction, whole cell; Lane 2: With IPTG induction, supernatant; Lane 3: Without IPTG induction, whole cell; Lane 4: Without IPTG induction, supernatant; Lane 5: Wild-Type

Melanin Synthesis

Achievement:

1. Successfully produce melanized bacteria

Process:

    After successfully synthesizing tyrosinase, the precursor Tyr as well as the cofactor Cu²⁺ were added into culturing medium LBYT. After overnight incubation, we observed that the medium turned dark with brown and diffusible pigments gradually accumulated for days. Besides liquid culture, the bacteria were also cultured in agar plates with the addition of Tyr and Cu²⁺, and it darkened as expected. Function test experiments were conducted to verify the identity of pigments in both solid and liquid mediums and the result are mentioned below.

(A)

(B)

Fig. 17. Melanin production in E. coli DH5α
(A) Melanin production in LBYT Agar Plate; (B) Melanin production in M9Y2 medium (centrifuged cells)

Production Optimization

Achievement:

1. Selected a suitable strain and its best culturing medium
2. Determined 37℃ is the better culturing temperature compared to 30℃
3. Determined the best concentration of cofactor Cu²⁺
4. Determined the effect of the transporter protein of precursor Tyr

Process:

    Experiments were conducted to reach a better production of melanin. To maximize production of melanin, we cultured the two strains in two kinds of medium, LBYT, and M9Y2, respectively. Melanin production was measured at OD₄₀₀ and the result is depicted below.

Table 2. Relative melanin production under different cultule conditions

Fig. 18. Culture condition and result

    Tyrosinase, the enzyme that catalyzes the oxidation of Tyr, has a cofactor Cu²⁺[10]. However, the culturing concentration of cofactor varied in previous studies. To determine the best concentration of the cofactor, we cultured bacteria with the melA gene and the addition of Cu²⁺ when OD₆₀₀ reached 0.6. The result is shown in Fig. 19 (A).

    Since some previous studies showed that melanin has a better production under the temperature of 32℃, we compared the efficiency of melanin production under 30℃ and 37℃. The result is presented in Fig. 19 (B).

(A)
(B)

Fig. 19. Optimization of the Cu²⁺ concentration (A) and temperature (B) for in vivo melanin production in E. coli DH5α

    As Fig. 19 shows, melanin production was better when cultured in 0.5 mM Cu²⁺ and 37℃.

    Tyrosine (Tyr) serves as an essential ingredient in melanin production. Tyrosine transporter protein (TyrP) was added into the pathway to import Tyr. Theoretically, a larger amount of Tyr is able to enter the cell with the addition of TyrP, and it was therefore expected to reach a higher production of melanin. The above experiment was conducted to verify the effectiveness of TyrP.

Fig. 20. Relative melanin production of E. coli with and without TyrP

    As Fig. 20 reports, melanin production increased after the addition of TyrP.

    The UV experiment was conducted to verify the effectiveness of synthesized melanin. Melanized bacteria, as well as non-melanized bacteria, were exposed to UV-B for 10 minutes and the CFU result is shown below. More information about how the experiment was conducted can be found in Measurement page.

(A)

(B)

Fig. 21. Bacteria under exposure of UV-B (A) Survival rate (B) CFU comparison.

GABA

GABA Production

Achievement:

1. GABA production with dual plasmids (melA and gadB)
2. Optimization of the substrate for in vivo GABA production

Process:

        Since our goal is to reach crucial substances biosynthesis in space, it is important to ensure the ability of Se coli factory. We chose GABA as a verification of our concept. After successfully constructing and transforming the plasmid containing gadB gene, the bacteria were cultured with PLP and glutamic acid. We measured GABA production with an OD meter after dyeing the cells. More information about how the production of GABA was measured can be found in Experiments page.

    The dual plasmids (melA and gadB) were transformed into the E. coli DH5α afterwards to make sure selenomelanin and GABA are produced simultaneously. Moreover, there were two different sets of backbones. We tested the production under two different plasmids, and the result showed that they both had a great ability for GABA synthesis.

Fig. 22. GABA production in the dual plasmid bacteria.

    There are two pathways that lead to two different substrates to synthesize GABA, which are glutamic acid and MSG respectively. The production of GABA was measured to determine the substrate used in further experiments, and the result is shown in Fig. 23.

Fig. 23. GABA production in substrate glutamic acid and PLP

    As shown above, GABA production did not show significant difference between adding glutamic acid and MSG. While the price of MSG is much lower than glutamic acid, so it is more economical to convert MSG into GABA in industry.

Selenomelanin

Selenomelanin Function Test

Achievement:

1. Successfully produced GABA with Se coli

Process:

    After successfully producing Sec and melanin, selenomelanin production was conducted. It had the same culturing condition and engineered bacteria as melanin production process at first, while Sec synthesized through in vitro tRNA pathway was added into the medium following the indication of previous study[11]. It was suggested that Sec was fed at the same concentration as L-DOPA. In addition, the amount of L-DOPA was estimated by the conversion of OD₄₀₀ value from previous experiment data to mass concentration.

    As the results shown below, Se coli had the best radiation tolerance towards UV irradiation exposure for an hour (12 W, UV-C) among the three groups. 17.9% of Se coli survived, while only 5% of melanized bacteria and 3.2% of non-melanized bacteria survived respectively. In general, selenomelanized bacteria (Se coli) is 3.6 times more tolerant to radiation than melanized-bacteria, and 6 times than non-melanized ones. This indicates the success of selenomelanin biosynthesis in Se coli.

(A)

(B)

Fig. 24. Bacteria under exposure of UV-C (A) Survival rate (B) CFU comparison

Fig. 25. GABA production in Se coli

    GABA-synthesizing Se coli was placed under UV irradiation as well as non-melanized and melanized bacteria to verify if selenomelanin protects microorganisms from radiation effectively. As the result indicates (Fig. 25), even though the amounts of GABA decrease because of melanin and selenomelanin production, the efficacy of GABA synthesis is still satisfying. This demonstrated the excellent radiation tolerance of GABA-synthesizing Se coli.