Overview

    The ultimate goal of our project is to engineer E. coli to produce selenomelanin, where selenocysteine (Sec) is the main precursor. Throughout the project, we have conducted design, build, test, learn iterations to determine the best pathway of Sec synthesis. To illustrate this, we separated our engineering success into three sections: tRNA synthesis, Enzyme synthesis, and Sec synthesis (Fig. 1).

Fig. 1. The three sections of engineering success

tRNA Synthesis

    Two ways of tRNA synthesis were designed, which are in vitro synthesis and in vivo overexpression. Though only the in vitro pathway has succeeded, two pathways will still be demonstrated in DBTL cycles to help the future iGEM teams (Fig. 2).

Fig. 2. DBTL cycles of tRNA synthesis: The left is for in vitro synthesis and the right is for in vivo overexpression.

In vitro tRNA Synthesis

Cycle I: Construction of tRNA^Ser and tRNA^Sec

Design:

    Two tRNA variants (Fig. 3), tRNA^Ser and tRNA^Sec, were constructed in the first cycle. According to a paper, tRNA^Ser has the best performance in aminoacylation of Ser to tRNA[1], while tRNA^Sec is the variant that is used in native Sec synthesis pathway[2]. To synthesize tRNA^Ser and tRNA^Sec, the tRNA genes must be constructed. The sequences with T7 promoters were ordered from IDT, cloned into TA vectors, and transformed into E. coli DH5α. Then, the target sequences were amplified through PCR before being condensed. At last, agarose gel electrophoresis and sequencing would be conducted.

Fig. 3. The sequences of tRNA^Ser and tRNA^Sec

Build:

    The synthesis process was conducted by following the standard procedure in our lab (see Experiments page).

Test:

    Blue-white screening, colony PCR, and gene sequencing were used to examine the results for ligation and transformation. As the results showed, the construction of tRNA^Ser and tRNA^Sec were both successful.

Learn:

    We learned that two variants of tRNAs might not be enough for comparison of the abilities for Sec Synthesis. Hence, another tRNA variant was introduced in the next cycle.

Cycle II: Construction of tRNA^UTuX

Design:

    The third tRNA variant (Fig. 4), tRNA^UTuX, is mutated from tRNA^Sec. According to papers, it has better performance in Sec synthesis than tRNA^Sec[3]. Hence, in the second cycle, tRNA^UTuX was constructed with the same strategy as the construction of tRNA^Ser and tRNA^Sec.

Fig. 4. The sequence of tRNA^UTuX

Build:

    The gene fragment of tRNA^UTuX variant underwent several reconstructions. At last, more colonies were picked for sequencing.

Test:

    After more colonies of E. coli with tRNA^UTuX were sent for sequencing, the tRNA^UTuX sequence finally matched the target one. Thus, we moved forward to conducting the third cycle: tRNA Synthesis.

Learn:

    The potential reasons for the previous failures of the tRNA^UTuX construction were concluded as follows. First, it is not a native tRNA sequence in E. coli. Second, it has a more complex sequence. Hence, the success of tRNA^UTuX construction required more steps.

Cycle III. tRNA synthesis

Design / Build:

    To obtain one of the main substrates of Sec synthesis, T7 RiboMAX^TM Express Large Scale RNA Production System (from Promega Corporation) was used to synthesize tRNA in vitro.

Test:

    NanoDrop and agarose gel electrophoresis were conducted to examine the results of tRNA synthesis. However, tRNA^Ser, tRNA^Sec,and tRNA^UTuX, all had two bands in the agarose gel electrophoresis results (Fig. 5).

Fig. 5. Confirmation of tRNA synthesis by agarose gel electrophoresis. The three tRNA species all appeared to have two bands
Lane1: tRNA^Ser; Lane2: tRNA^Sec; Lane3: tRNA^UTuX.

    To synthesize tRNA^Ser, tRNA^Sec, and tRNA^UTuX with right size, we referred to several tRNA reanneling protocols. After synthesizing tRNAs with the aforementioned commercial kit, the tRNA products were reannealed by diluting the sample, heated to break the hydrogen bond between the nucleotides, and cooled down. Finally, we derived the expected results (Fig. 6).

Fig. 6. Confirmation of tRNA synthesis by agarose gel electrophoresis
Lane1: tRNA^Ser; Lane2: tRNA^Sec; Lane3: tRNA^UTuX

Learn:

    After consultation and discussion, it was speculated that the high concentration of tRNA products might be the reason for the occurrence of two bands. If the concentration of unfolded tRNA solution is too high during the synthesis process, the nearby tRNAs would be more likely to anneal with each other. Thus, it would result in a larger size of the final products, which could be observed in the gel electrophoresis results.

In vivo tRNA Synthesis

Cycle I: Three-step construction

Design:

    An in vivo tRNA synthesis pathway is also designed, which could lead to more yields of tRNAs with proper post-transcriptional modifications. According to literatures[4], with lpp promoter (P_lpp) in the front and rrnC terminator (T_rrnC) in the back, the three tRNA variants, tRNA^Ser, tRNA^Sec, and tRNA^UTuX, can be overexpressed in vivo. Thus, we decided to construct the tRNA sequences into the TA vector at first, and then construct P_lpp in the 5’ end while T_rrnC in its 3’ end.

Build:

    E. coli DH5α is used as the host for transformation. The three DNA fragments, which were P_lpp, tRNA gene, and T_rrnC, were constructed into the plasmid one by one in the order: tRNA, T_rrnC, and P_lpp.

Test:

    Through colony PCR, the construction of tRNAs was successful. Also, the construction of T_rrnC succeeded after we tried a few times by increasing the efficiency of the transformation. Unfortunately, we failed to obtain the clone with the correct sequences.

Learn:

    Since we had limited time to repeat the construction cycle, we decided to try the alternative way, which is to ligate three fragments at a time.

Cycle II: One-step construction

Design:

    The cloning method we previously used was too time-consuming, so another method was adopted: to create the different sticky ends of the three target DNA fragments by using restriction enzymes, then to ligate the fragments together by using the T4 ligase. The ligation product will be cloned into a TA vector (a high-copy vector) or pSAA (a low-copy vector).

Build:

    Before ligation, there are three fragments needed to be prepared:

1. tRNA gene sequence with EcoRI restriction site sticky end in the front and BamHI restriction site sticky end in the back
2. lpp promoter with KpnI restriction site sticky end in the front and EcoRI restriction site sticky end in the back
3. rrnC terminator with BamHI restriction site sticky end in the front and PstI restriction site sticky end in the back

    To prepare the first fragment, the plasmids already constructed with the tRNA gene sequence were digested with EcoRI and BamHI. At the same time, lpp promoter and rrnC terminator were obtained by using nucleotide synthesis with the corresponding two DNA strands. The respective DNA synthetic sequences were aligned and created the particular dsDNA fragments with the sticky ends on two sides.

Test:

    After ligating the three fragments simultaneously, agarose gel electrophoresis was conducted to check the result. As the results showed, the ligation products appeared to have various sequences; moreover, it was difficult to determine the target sequence.

Learn:

    Even though a lot of parameters were modified during the experiments, for instance, we adjusted the amount of DNA template and enzymes, or we even modified the gel purification method, the in vivo tRNA synthesis pathway still did not succeed.

    Moreover, limited time had forced us to give up on this pathway and use in vitro synthesized tRNAs as the primary substrate for the Sec in vitro synthesis experiment. Since in vivo transcription still has some advantages, such as the final products with high yield or the products with post-transcriptional modification, we hope to conduct it in the future. Moreover, other methods will be tried for this complex construction.

Enzyme Synthesis

Cycle: Expression of SerRS, SelA, and SelD protein

Design:

    To produce enough amounts of SerRS, SelD and SelA protein for Sec in vitro synthesis, SerRS, SelD and SelA in E. coli BW25113 were overexpressed respectively and purified through his-tag columns. The expressed proteins were examined by performing SDS-PAGE and the concentration was determined with NanoDrop for further uses in Sec synthesis.

Build:

    The enzymes were overexpressed with IPTG induction and conducted protein purification following standard procedure in our laboratory (see Experiments page).

Test:

    We took samples from every step of protein purification. Also, we conducted SDS-PAGE to ensure their purity and to avoid protein loss during purification. NanoDrop was also used to quantify the levels of the protein. As Fig. 7 shows, SerRS, SelD, and SelA were produced successfully.

Fig. 7. Confirmation of enzyme synthesis by western blotting
M: Marker; Lane 1: SerRS; Lane 2: SelD; Lane 3: SelA

Learn:

    Though there was little protein loss or impurity during purification, the total amount of SelA was relatively low from the desired amount for the following experiments. The cell growth was later measured by monitoring OD₆₀₀ value, and the result showed that the overexpression of SelA protein accumulated in bacteria inhibited the cell growth. In the future, we will alter several culturing factors, such as total culturing time and timing of IPTG induction to maximize protein production.

Sec Synthesis

Fig. 8. DBTL cycles of Sec synthesis

Cycle I: First attempt to synthesize Sec

Design:

    We referred to the protocols from various papers to synthesize Sec through the in vitro pathway. There are two steps left for Sec synthesis, which are aminoacylation[5] and conversion of seryl-tRNA to selenocysteinyl-tRNA[6].

Build:

    First, aminoacylation was conducted, in which the enzyme SerRS would make Ser bind with tRNA. Then, the enzymes SelD and SelA were utilized to convert seryl-tRNA into selenocysteinyl-tRNA.

Test:

    To examine the products of the reaction, Urea PAGE and HPLC were used as the measuring methods. As for Urea PAGE, aminoacyl-tRNA should be distinguished from tRNA owing to size and polarity differences. However, the bands of Urea PAGE appeared to be vague. As the HPLC results showed, we did not successfully synthesize Sec.

Learn:

    Since Urea PAGE was conducted at room temperature and the running time was too long, we thought that the tRNA samples would degrade when moving to the bottom of the gel.

    According to the HPLC result, we troubleshot possible reasons for the failures, and determined that the loss of protein activity might be a significant reason.

Cycle II: Second attempt to synthesize Sec

Build:

1. Urea PAGE:
       From the previous experience, we decided to shorten the running time and gel length, and run the gel in a refrigerator.
2. HPLC:
       This time, we ensured that the proteins were fresh enough by using them right after they were synthesized and purified.

Test:

    To examine the result of the adjusted methods, we conducted Urea PAGE and HPLC.

1. Urea PAGE:
       The bands of seryl-tRNAs and selenocysteinyl-tRNAs had upshifted compared to the band of the tRNA. The results showed that the amino acid was successfully charged to the tRNA (Fig. 9 and 10).


Fig. 9. 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. 10. 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

2. HPLC:
       As the results showed, the peaks around 3.5 min, 4.5 min, and 10.5 min were similar to those of the HPLC results of Sec standard (Fig. 11).
    In this result, the peaks around 3.5 min, 4.5 min, and 10.5 min are similar to those of the HPLC result of Sec standard (Fig. 6).

(A)



(B)



Fig. 11. Confirmation of Sec by HPLC (A) Sec synthesized by tRNA^Ser; (B) Sec synthesized by tRNA^UTuX

Learn:

    We learned that the enzyme activity would be a decisive element in influencing the results of in vitro pathway; moreover, the property of tRNA samples should be considered when running Urea PAGE.

    In the future, designing examining methods will be necessary to measure the enzyme activity better. For instance, we consider designing a controlled experiment by adding inactivated SelA and SelD in the experiment of seryl-tRNA to selenocysteinyl-tRNA.

Conclusions

    As the results showed, two of the three selected tRNA variants, tRNA^Serand tRNA^UTuX were able to synthesize Sec by using in vitro synthesis. With the DBTL cycles, it was proved that the 21^st amino acid, selenocysteine (Sec), can be derived from the enzyme overexpression in E. coli and in vitro tRNA synthesis. We provide a new methodology to obtain this special, but important amino acid for further application.