iGEM 2022 — Team ncku-tainan

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2022/5/30

2022/6/6

2022/6/13

2022/6/20

2022/6/27

2022/7/4

2022/7/11

2022/7/18

2022/7/25

2022/8/1

2022/8/8

2022/8/15

2022/8/22

2022/8/29

2022/9/5

2022/9/12

2022/9/19

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| Wet   1. Construct plasmid 2. Design primers for tRNA 3. Construct the sequences of tRNA  1. Construct the pSU-cysK plasmid 2. Design primers for tRNASer, tRNASec, and tRNAUTuXconstruction 3. Construct the sequences of tRNASer and tRNASec with T7 promoter into TA vector  Dry   1. Hardware brainstorming 2. Meet with experts 3. Study the basic knowledge of modeling  1. Hardware brainstorming 2. Meet with experts to ask for advices on the hardware ideas 3. Study the enzyme kinetics and other basic knowledge of modeling  HP   1. Smart community workshop 2. ARS preparation 3. Fortnightly and talk show preparation  1. Participate in Smart community workshop 2. ARS preparation 3. Prepare for the second episode of the fortnightly and the talk show 4. Discuss collaboration details with NCHU |
| Wet   1. Construct plasmid 2. Design primers for tRNA 3. Construct the sequences  1. Construct the pSU-cysK plasmid 2. Design primers for tRNASer, tRNASec, and tRNAUTuX 3. Construct the sequences of tRNASer and tRNASec with T7 promoter into TA vector  Dry   1. Hardware brainstorming 2. Meet with experts 3. Search for information  1. Hardware brainstorming 2. Meet with experts to ask for advices on the hardware ideas 3. Search for useful model information regarding our project  HP   1. ARS preparation 2. Fortnightly and talk show preparation 3. Post team member introduction  1. ARS preparation 2. Prepare for the second episode of the fortnightly and the talk show 3. Post team member introduction on Instagram 4. Discuss collaboration details with NCHU |
| Wet   1. Construct plasmids 2. Perform PCR to copy tRNA sequences and condense the PCR results  1. Construct pSU-cysK-cysE\* and pSU-cysE\* plasmids 2. Perform PCR to copy tRNASer, tRNASec, tRNAUTuX DNA sequences on TA vector and condense the PCR results  Dry   1. Meet with experts 2. Research 3. Browse previous iGEM team Best Model  1. Meet with experts 2. Research on all kinds of microgravity simulators 3. Browse previous iGEM team Best Model winner teams' wiki 4. Learn wiki editting  HP   1. Logo design 2. Entrepreneurship design 3. ARS and talk show preparation  1. Logo design 2. Entrepreneurship design 3. ARS and talk show preparation 4. Education kit design |
| Wet   1. Sequencing of *cysE\** and *ydeD* 2. Check *cysE\** function 3. Synthesize RNA  1. Do sequencing to check the structures of *cysE\** and *ydeD* 2. Check *cysE\** function by transforming pSU-cysE\* into *△cysE* and measure the OD value 3. Synthesize RNA *in vitro*  Dry   1. Meet with experts 2. Start building hardware 3. Design Wiki page  1. Meet with experts 2. Start building UV stage and designing clinostat 3. Determine the main colors of Wiki page  HP   1. Promotion video preparation 2. Logo design 3. ARS and talk show preparation  1. Promotion video preparation 2. Logo design 3. ARS and talk show preparation 4. Education kit, literature, entrepreneurship, activity design |
| Wet   1. Purify SerRS, SelA and SelD 2. Synthesize RNA  1. Purify SerRS, SelA and SelD with His-tag 2. Synthesize RNA  Dry   1. Software idea discussion 2. Building hardware 3. Reading papers  1. Software idea discussion 2. Build the first prototype of UV stage 3. Reading papers regarding the melanin pathway 4. Determine the Wiki page structure and design the layout  HP  1. Logo design 2. Collaboration discussion 3. Literature, activity design  1. Logo design 2. Collaboration discussion 3. Literature, activity design 4. Talk show preparation |
| Wet   1. Transform *ydeD* 2. Measure the melanin production 3. Construct backbones  1. Transform *ydeD* into MG1655 and *△iscS* 2. Measure the melanin production under different temperatures and mediums 3. Construction of different backbone of plasmids  Dry   1. Build clinostat 2. Consult Landing AI 3. Read papers for modeling  1. Build clinostat 2. Consult Landing AI about software design 3. Read papers regarding the selenocysteine pathways 4. Design Wiki layout  HP   1. Audiobook design 2. Discuss collaboration 3. Visit National Academy of Taiwan  1. Audiobook design 2. Discuss the collaboration details with other schools 3. Visit National Academy of Taiwan 4. Discuss entrepreneurship details with Gran Systems |
| Wet   1. Transform pcysK-cysE\* 2. Reanneal the synthesized RNA 3. Run SDS-PAGE  1. Transform pcysK-cysE\* into MG1655 and *△iscS* with and without *ydeD* 2. Reanneal the synthesized RNA 3. Run SDS-PAGE to analyze temperature effects on protein expression of *melA* 4. Test the survival rate of the bacteria with melanin under UV radiation  Dry   1. Build clinostat 2. Software design 3. Read papers  1. Build clinostat and encounter some difficulties 2. Software design 3. Read papers regarding the selenocysteine pathway 4. Start editing the Wiki templates  HP   1. Audiobook design 2. Find sponsorship 3. Entrepreneurship, education kit design  1. Audiobook design 2. Find sponsorship 3. Entrepreneurship, education kit design 4. Talk show preparation |
| Wet   1. Compare the growth rate 2. Condense SerRS, SelA and SelD  1. Compare the growth rate of MG1655 and *△iscS* with constructed plasmids 2. Condense SerRS, SelA and SelD; run SDS-PAGE with Western blotting and coomassie blue staining  Dry  1. Discuss about microfluidic chip 2. Start constructing the software 3. Modeling the melanin pathway  1. Discussion about the idea of microfluidic chip 2. Start constructing the software 3. Modeling the melanin pathway 4. Wiki page editing and art design  HP   1. Promotion video preparation 2. Discuss the collaboration details with other schools 3. Literature, slides and activity design  1. Promotion video preparation 2. Discuss the collaboration details with other schools 3. Literature, slides and activity design 4. Talk show 2.0 preparation |
| Wet   1. Aminoacylation of tRNA and serine 2. Conversion of seryl-tRNA 3. Compare the function  1. Aminoacylation of tRNA and serine 2. Conversion of seryl-tRNA to selenocysteinyl-tRNA 3. Compare the function among pSB4KI-Ptrc-melA and pSB4KI-Ptrc-melA-B001  Dry   1. Learn basic knowledge and visit NHRI 2. Present our modeling idea to professors 3. Use AI platform for software model training  1. Learn the basic knowledge of microfluidic chip and visit NHRI to fabricate our first version of microfluidic chips 2. Present our modeling idea to professors and discover some problems to be solved 3. Use AI platform for software model training 4. Wiki page editing and art design  HP   1. Audiobook recording 2. Promotion video preparation 3. Wiki page, IHP content discussion  1. Audiobook recording 2. Promotion video preparation 3. Wiki page, IHP content discussion 4. Talk show 2.0 preparation |
| Wet   1. Test different sodium selenite concentrations 2. Deacylate selenocysteine 3. Transform *gadB*  1. Culture pcysK-cysE\*, pPompA-ydeD/MG1655 in different sodium selenite concentrations.- Deacylate selenocysteine from tRNA    - Transform *gadB* to *E. coli* to produce GABA    - Compare the function of melanin production between DH5a, MG1655, BL21, and W3310  Dry   1. Design the second prototype of UV stage 2. Software model training 3. Learn Copasi and MATLAB  1. Design the second prototype of UV stage 2. Continue with software model training 3. Learn how to use Copasi and MATLAB for pathway simulation 4. Build our melanin pathway model 5. Discuss hanging drop simulation 6. Wiki page editing and art design  HP   1. Audiobook recording 2. Promotion video preparation 3. Wiki page, IHP content discussion  1. Audiobook recording 2. Promotion video preparation 3. Wiki page, IHP content discussion 4. Literature design 5. Talk show 2.0 preparation |
| Wet   1. Compare melanin production 2. Test different substrates for GABA production 3. Conversion of seryl-tRNA  1. Compare melanin production with and without TyrP between DH5a, MG1655, and BL21 2. Measure the production of GABA under different substrates. 3. Conversion of seryl-tRNA to selenocysteyl-tRNA  Dry   1. Microfluidic chip fabrication 2. Software coding 3. Selenocysteine pathway model  1. Visit NHRI to fabricate the microfluidic chip with different well diameter for future testing and improvement 2. Start coding for our software 3. Build our two selenocysteine pathway model 4. Finish the art design of the Wiki main page, Team page  HP   1. Audiobook recording 2. Promotion video preparation 3. Wiki page, IHP content discussion  1. Audiobook recording 2. Promotion video preparation 3. Wiki page, IHP content discussion 4. Literature design 5. SDG poster design 6. Talk show 2.0 preparation |
| Wet   1. UV experiments 2. Construction terminator and promoter 3. Remove *KmR*  1. Test the melanin function by UV experiments 2. Constuction of *rrnC* terminator and *lpp* promoter 3. Remove *KmR* from *ΔtyrA*  Dry   1. Survival rate experiment 2. Taguchi experiments 3. Medium exchange model  1. Survival rate experiment in MerStage 2. Finish Taguchi experiments 3. Learn how to use Ansys Fluent to simulate medium exchange in the microfluidic chip 4. Wiki animation coding  HP   1. Talk show 2.0 2. Discuss with companies 3. Collaboration with other schools  1. Talk show 2.0 2. Discuss with companies 3. Collaboration with other schools |
| Wet   1. Dual plasmids GABA production 2. Deacylation 3. Amino acid purification  1. Measure the production of GABA from dual plasmids 2. Deacylation with nuclease S1 and Tris-HCl 3. Amino acid purification 4. Confirm the production of selenocysteine by HPLC  Dry   1. Survival rate experiment 2. Confirmation experiments 3. Cell aggregation model discussion with experts  1. Survival rate experiment in MerStage 2. Finish confirmation experiments for Taguchi methods 3. Discussion with Prof. Wei about nutrient consumption rate calculation and cell aggregation model 4. Wiki animation coding  HP   1. Promotion video 2. Discuss with companies 3. Collaboration with other schools  1. Finich making promotion video 2. Discuss with companies 3. Collaboration with other schools |
| Wet   1. Protocol adjustment 2. Urea PAGE 3. Confirm YdeD function  1. Adjustment of digestion and ligation protocol 2. Apply Urea PAGE 3. Use CFU to confirm YdeD function 4. Confirm the production of selenocysteine by HPLC  Dry   1. Discuss different fabrication methods 2. Survival rate experiment 3. Cell aggregation model  1. Discussion with Prof. Juang about different chip fabrication methods 2. Survival rate experiment in MerStage 3. Finish construction of cell aggregation model 4. Wiki debug  HP   1. Workshop preparation 2. Wiki page writing 3. Wiki page adjustments  1. Workshop preparation 2. Wiki page writing 3. Wiki page adjustments 4. Literature design |
| Wet   1. Produce selenomelanin 2. Urea PAGE  1. Start to produce selenomelanin and compare it with melanin 2. Urea PAGE method adjustment  Dry   1. Survival rate experiment 2. MerStage optimization 3. Melanin pathway model  1. Survival rate experiment in MerStage 2. MerStage optimization 3. Finish melanin pathway experiments and model 4. Wiki page content writing and coding  HP   1. Workshop preparation 2. Wiki page writing 3. Wiki page adjustments  1. Workshop preparation 2. Wiki page writing 3. Wiki page adjustments |
| Wet   1. Test selenomelanin function 2. Wiki page writing  1. Test the function of selenomelanin 2. Wiki page writing  Dry   1. Wiki page writing  1. Wiki page writing  HP   1. Workshop 2. Wiki page writing 3. Wiki page adjustments  1. Workshop 2. Wiki page writing 3. Wiki page adjustments |
| Wet  1. Wiki page writing 2. Wiki page adjustments  1. Wiki page writing 2. Wiki page adjustments  Dry   1. Wiki page writing 2. Wiki page adjustments 3. Wiki debug  1. Wiki page writing 2. Wiki page adjustments 3. Wiki debug  HP   1. Wiki page writing 2. Wiki page adjustments  1. Wiki page writing 2. Wiki page adjustments |

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HP

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![](https://static.igem.wiki/teams/4171/wiki/engineering-f.png)

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).

![](https://static.igem.wiki/teams/4171/wiki/engineering-fig-1.png)

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).
![](https://static.igem.wiki/teams/4171/wiki/engineering-fig-2.png)

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 tRNASer and tRNASec

Design:

    Two tRNA variants (Fig. 3), tRNASer and tRNASec, were constructed in the first cycle. According to a paper, tRNASer has the best performance in aminoacylation of Ser to tRNA[1], while tRNASec is the variant that is used in native Sec synthesis pathway[2]. To synthesize tRNASer and tRNASec, 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.

![](https://static.igem.wiki/teams/4171/wiki/engineerfig-3.png)

Fig. 3. The sequences of tRNASer and tRNASec

Build:

    The synthesis process was conducted by following the standard procedure in our lab (see [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) 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 tRNASer and tRNASec 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 tRNAUTuX

Design:

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

![](https://static.igem.wiki/teams/4171/wiki/engineerfig-4.png)

Fig. 4. The sequence of tRNAUTuX

Build:

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

Test:

    After more colonies of *E. coli* with tRNAUTuX were sent for sequencing, the tRNAUTuX 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 tRNAUTuX 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 tRNAUTuX construction required more steps.

Cycle III. tRNA synthesis

Design / Build:

    To obtain one of the main substrates of Sec synthesis, T7 RiboMAXTM 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, tRNASer, tRNASec,and tRNAUTuX, all had two bands in the agarose gel electrophoresis results (Fig. 5).
![](https://static.igem.wiki/teams/4171/wiki/engineerfig5.png)

Fig. 5. Confirmation of tRNA synthesis by agarose gel electrophoresis. The three tRNA species all appeared to have two bands
Lane1: tRNASer; Lane2: tRNASec; Lane3: tRNAUTuX.

    To synthesize tRNASer, tRNASec, and tRNAUTuX 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).

![](https://static.igem.wiki/teams/4171/wiki/engineerfig-6.png)

Fig. 6. Confirmation of tRNA synthesis by agarose gel electrophoresis
 Lane1: tRNASer; Lane2: tRNASec; Lane3: tRNAUTuX

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 (*Plpp*) in the front and *rrnC* terminator (*TrrnC*) in the back, the three tRNA variants, tRNASer, tRNASec, and tRNAUTuX, can be overexpressed *in vivo*. Thus, we decided to construct the tRNA sequences into the TA vector at first, and then construct *Plpp* in the 5’ end while *TrrnC* in its 3’ end.

Build:

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

Test:

    Through colony PCR, the construction of tRNAs was successful. Also, the construction of *TrrnC* 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) 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.

![](https://static.igem.wiki/teams/4171/wiki/fig-7-final.png)

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

![](https://static.igem.wiki/teams/4171/wiki/engineering-fig-8.png)

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).
![](https://static.igem.wiki/teams/4171/wiki/engineerfig9.png)

Fig. 9. Confirmation of the result after aminoacylation and conversion of seryl-tRNA to selenocysteinyl-tRNA by Urea PAGE
Lane1: tRNASer; Lane2: seryl-tRNASer; Lane3: selenocysteinyl-tRNASer;
Lane4: tRNASec; Lane5: seryl-tRNASec; Lane6: selenocysteinyl-tRNASec

![](https://static.igem.wiki/teams/4171/wiki/engineerfig10.png)

Fig. 10. Confirmation of the result after aminoacylation and conversion of seryl-tRNA to selenocysteinyl-tRNA by Urea PAGE
Lane1: tRNAUTuX; Lane2: seryl-tRNAUTuX; Lane3: selenocysteinyl-tRNAUTuX

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)

![](https://static.igem.wiki/teams/4171/wiki/engineerfig-11-a-png.png)

(B)

![](https://static.igem.wiki/teams/4171/wiki/engineerfig-11-b-png.png)

Fig. 11. Confirmation of Sec by HPLC (A) Sec synthesized by tRNASer; (B) Sec synthesized by tRNAUTuX

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, tRNASerand tRNAUTuX were able to synthesize Sec by using *in vitro* synthesis. With the DBTL cycles, it was proved that the 21st 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.

References

[1] Asahara, H., Himeno, H., Tamura, K., Nameki, N., Hasegawa, T., Shimizu, M. (1994). Escherichia coli seryl-tRNA synthetase recognizes tRNASer by its characteristics tertiary structure. *Journal of Molecular Biology*. 236(3):738-748. doi:10.1006/jmbi.1994.1186
[2] Escherichia sp. E10V4 tRNA-Sec | URS00002F9CDD. rnacentral.org. Accessed September 26, 2022. https://rnacentral.org/rna/URS00002F9CDD/2478971
[3] Fu, X., Crnković, A., Sevostyanova, A., Söll, D. (2018). Designing seryl‐ tRNA synthetase for improved serylation of selenocysteine tRNA s. *FEBS Letters*, 592(22), 3759-3768. doi:10.1002/1873-3468.13271
[4] Meinnel, T., Mechulam, Y., Fayat, G., Blanquet, S. (1992). Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and *E. coli* methionyl-tRNA synthetases. *Nucleic Acids Research*. 20(18):4741-4746. doi:10.1093/nar/20.18.4741
[5] Walker, S. E., Fredrick, K. (2008). Preparation and evaluation of acylated tRNAs. *Methods*. 44(2):81-86. doi:10.1016/j.ymeth.2007.09.003
[6] Forchhammer, K., Böck, A. (1991). Selenocysteine synthase from Escherichia coli. Analysis of the reaction sequence. *Journal of Biological Chemistry*, 266(10), 6324-6328. doi:10.1016/s0021-9258(18)38121-3

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![](https://static.igem.wiki/teams/4171/wiki/result.png)

[Overview](#page1)
[tRNA Synthesis](#page2)
[Enzyme Synthesis](#page3)
[Sec Synthesis](#page4)
[Conclusions](#page5)
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![](https://static.igem.wiki/teams/4171/wiki/expereiment-for-2nd-freeze.png)

1. Media Preparation

LB (Lysogeny Broth)

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| Materials  Equipment:   1. Glass bottle (for LB broth) or Erlenmeyer flask (for agar medium) 2. Magnetic stirrer 3. Magnetic stirring bar 4. Plastic measure jug 5. Measure cylinder 6. Spoon 7. Petri dish (for agar medium) 8. Aluminum foil 9. Electronic balance 10. Weighing paper or weighing bowl 11. Pipette and pipette tips   Consumables:   1. DW (distilled water) 2. Tryptone 3. Yeast extract 4. NaCl 5. 10 M NaOH 6. Agar (for LB agar) 7. Antibiotics (for selection plate) | Protocol   1. Prepare the mixture as the following in a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.     |  |  |    | --- | --- |    | Components | Volume |    | DW | 98 mL |    | Tryptone | 1 g |    | Yeast Extract | 0.5 g |    | NaCl | 1 g |    | 10 M NaOH | 20 μL |    | Total Volume | 100 mL |    | Agar (for LB agar) | 1.5% | 2. Mix well, and pour the mixture into the bottle or flask and autoclave. Notes: For agar medium, pour the mixture into the petri dish and dry the plates.   For selection plates, add antibiotics as the following before pouring the mixture into the petri dish:   1. Kanamycin: 100 μL/100 mL media 2. Chloramphenicol: 50 μL/100 mL media 3. Ampicillin: 100 μL/100 mL media |

SOB (Super Optimal Broth)

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| Materials  Equipment:   1. Flask 2. Magnetic stirrer 3. Magnetic stirring bar 4. Plastic measure jug 5. Measure cylinder 6. Spoon 7. Aluminum foil 8. Electronic balance 9. Weighing paper or weighing bowl 10. Pipette and pipette tips   Consumables   1. DW (distilled water) 2. Tryptone 3. Yeast extract 4. 5 M NaCl 5. 3 M KCl | Protocol   1. Prepare the mixture as the following in a jar with a magnetic stirring bar inside and place it on the magnetic stirrer.     |  |  |    | --- | --- |    | Components | Volume |    | DW | 96 mL |    | Tryptone | 2 g |    | Yeast Extract | 0.5 g |    | 5 M NaCl | 0.2 mL |    | 3 M KCl | 83 μL |    | Total Volume | 100 mL | 2. Mix well, and pour the mixture into the flask and autoclave. |

SOC

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| Materials:  Equipment:      Pipette and pipette tips  Consumables:   1. SOB medium in a glass bottle 2. 1 M MgCl2 3. 1 M MgSO4 4. 20% glucose | Protocol  Add chemicals as the following to a bottle of 100 mL SOB (autoclaved).   |  |  | | --- | --- | | Components | Volume | | 1 M MgCl2 | 96 mL | | 1 M MgSO4 | 2 g | | 20% glucose | 0.5 g | |

M9

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| Materials  Equipment:      Pipette and pipette tips  Consumables:   1. M9 salts (5X) 2. 20% glucose 3. 1 M MgSO4 4. 1 M CaCl2 5. H2O | Protocol   1. Add chemicals as the following to a bottle.     |  |  |    | --- | --- |    | Components | Volume |    | M9 salts (5X) | 20 mL |    | 20% glucose | 2 mL |    | 1 M MgSO4 | 200 μL |    | 1 M CaCl2 | 10 μL |    | H2O | 78 mL |    | Total Volume | 100 mL | 2. Mix well, and pour the mixture into the flask and autoclave. |

2. GABA Production

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Centrifuge 4. Hot plate 5. OD meter   Consumables:   1. 0.4 M Boric acid buffer (pH 10) 2. 6% Phenol 3. 6% Sodium hypochlorite (NaClO) 4. Overnight cultured bacteria (with plasmids encoded *gadB* gene) | Protocol   1. Add 1.5 mL overnight cultured bacteria to a 1.5 mL microfuge tube. 2. Centrifuge at 14000 rpm for 3 minutes. 3. In a 2 mL eppendorf add all the components below in order, making sure that phenol is being added in the fume hood.     |  |  |    | --- | --- |    | Components | Volume |    | 0.4 M Boric acid buffer (pH 10) | 100 μL |    | Supernatant of bacteria | 10 μL |    | 6% Phenol | 100 μL |    | 6% Sodium hypochlorite (NaClO) | 100 μL | 4. After adding all the components, place the microfuge at room temperature for 5 minutes. 5. Put it onto the hot plate at 100℃ for 10 minutes. 6. Load 200 μL of the sample and the standard into 96 wells. 7. Test the OD value at 630 nm by OD meter and draw the standard curve to later calculate the concentration of GABA. |

3. Survival Rate Test

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Culturing plate 4. OD meter   Consumables:   1. LB medium 2. Overnight cultured bacteria (with plasmids encoded *gadB* gene and *melA* gene) | Protocol  The experiment was divided into three parts: UV light exposure, dilution and dropping.  UV light exposure   1. Expose the plates which have bacteria colonies to UV light for 5 minutes. 2. Wash down the bacteria colonies with LBYT medium. 3. Adjust the OD values of LBYT medium containing bacteria to 1.   Dilution  Dilutes the samples 1~10-6 times   |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | | Concentration | 1 | 10-1 | 10-2 | 10-3 | 10-4 | 10-5 | 10-6 | | LB medium (μL) | 0 | 180 | 180 | 180 | 180 | 180 | 180 | | Sample (μL) | 200 | 20 | 20 | 20 | 20 | 20 | 20 |   Dropping  Drop 5 μL of each sample onto the agar plates, and culture them overnight. |

4. PCR

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| Materials  Equipment:   1. PCR tubes 2. Ice bucket 3. Thermocycler 4. Pipette and pipette tips   Consumables:   1. Sterilized ddH2O 2. 10X PCR buffer for Ex Taq 3. 2 mM dNTP 4. Forward and reverse primer (10 μM) 5. Ex Taq polymerase | Protocol   1. Add the following chemicals to the PCR tube on ice:     |  |  |    | --- | --- |    | Components | Volume (μL) |    | Template DNA | 1 |    | 10X PCR buffer for Ex Taq | 5 |    | 2 mM dNTP | 5 |    | Primer (forward and reverse) | 2 |    | Ex Taq polymerase | 0.1 |    | Sterilized ddH2O | 36.9 |    | Total Volume | 50 | 2. Gently mix the PCR solution and centrifuge briefly. 3. Transfer the PCR tubes to a thermocycler.     |  |  |  |    | --- | --- | --- |    | Step | Temperature (℃) | Time (second) |    | Initial denaturalization | 94 | 300 |    | 25 - 35 cycles | 94 (denaturation) | 30 |    | 55 (annealing) | 30 |    | 72 (extension) | 60 |    | Final extension | 72 | 600 |    | Hold | 16 | infinity | |

5. Restriction Enzymes Digestion

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| |  |  | | --- | --- | | **Restriction Enzymes** | **Buffer (Buffer depends on the restriction enzyme being used)** | | EcoRI | r2.1 (10X) - NEB || PstI-HF | rCutSmart (10X) - NEB | | KpnI-HF | rCutSmart (10X) - NEB | | BamHI | r2.1 (10X) - NEB | | |
| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Ice bucket 4. Incubator (37℃)   Consumables:   1. Restriction enzymes 2. 10X Buffer (depends on its restriction enzymes) 3. DNA sample 4. MQ or ddH2O (for single digestion) | Protocol   1. On the ice, add all the following components.     Double Digestion:     |  |  |    | --- | --- |    | Components | Volume |    | DNA | 43 μL |    | 10X Buffer | 5 μL |    | Restriction enzyme 1 | 1 μL |    | Restriction enzyme 2 | 1 μL |    | Total Volume | 50 μL |     Single Digestion (Structure Check):     |  |  |    | --- | --- |    | Components | Volume |    | Plasmid DNA | 2 μL |    | 10X Buffer | 1.5 μL |    | Restriction enzyme | 0.5 μL |    | MQ or ddH2O | 11 μL |    | Total Volume | 15 μL | 2. Mix gently and incubate for 3 hours for double digestion or 1 hour for single digestion at 37℃. *Note: Incubation time varies along the total volume of the reaction.* |

6. Ligation

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Ice bucket   Consumables:   1. T4 DNA ligase (Takara Bio) 2. 10X T4 DNA ligase buffer (Takara Bio) | Protocol   1. Add the following chemicals to the PCR tube on ice:     |  |  |    | --- | --- |    | Components | Volume |    | Vector | 2 μL |    | Insert | 6 μL |    | 10X buffer | 1 μL |    | T4 ligase | 1 μL |    | Total Volume | 10 μL | 2. Gently mix the reaction and centrifuge. 3. Incubate at 4℃ overnight. 4. Keep the DNA solution in the -20℃ freezer. |

7. Transformation

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Ice bucket 4. Centrifuge   Consumables:   1. Competent cell 2. IPTG/X-Gal 3. Plasmid DNA or Ligation product 4. Lysogeny broth (LB) 5. LB agar with Ampicillin (Amp) | Protocol   1. Thaw frozen competent cells on ice. 2. Add 10 μL ligation product or 1 μL plasmid DNA per 50 μL tube of competent cells, and quickly flick the tube several times. 3. Keep tubes on ice for 30 minutes. 4. Heat-shock the cells for 45 seconds in a water bath at exactly 42℃. 5. Immediately place the tubes on ice for 1 minute. 6. Add 1 mL LB medium to each transformation reaction and incubate at 37℃ for 2 hours at 180 rpm in a shaking incubator. 7. Centrifuge for 4 minutes at 4℃ and 5000 xg. 8. Discard the supernatant (about 800 μL to 1 mL) and keep a 200 μL medium. 9. Add 50 μL IPTG/X-Gal (IPTG:X-Gal; 1:4 premix), and resuspension. 10. For each transformation reaction, take 100 μL transformation product to LB agar plate with Amp and spread evenly. 11. Incubate the plates at 37℃ for 12-14 hours or overnight. |

8. tRNA Synthesis

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| Materials  Equipment:   1. PCR tubes 2. Pipette and pipette tips 3. Ice bucket 4. Centrifuge   Consumables:   1. Nuclease free water 2. Ribomax 2X buffer 3. Enzyme mix 4. DNase 5. Isopropanol 6. Phenol (pH 4.3) 7. NaOAC 8. Chloroform + isoamyl alcohol (24:1 premix) 9. 75% EtOH 10. DEPC-treated water | Protocol   1. On the ice, add all components in a PCR tube, making up to 20 μL volume reaction.     |  |  |    | --- | --- |    | Components | Volume |    | DNA template (1 μg/μL) | 1 μL |    | Nuclease-free water | 7 μL |    | Enzyme mix | 2 μL |    | Ribomax 2X buffer | 10 μL |    | Total Volume | 20 μL | 2. Gently mix the PCR reactions and centrifuge briefly. 3. Synthesis reaction for 3 hours at 37℃. 4. Reanneal tRNA for 20 minutes at 70℃. 5. Cold down for 30 minutes at room temperature. 6. Add 2 μL DNase to degrade DNA template for 15 minutes at 37℃. 7. Add phenol 52.5 μL and chloroform + isoamyl alcohol 10.5 μL in each tube 8. Vortex 1 minute, and Centrifuge for 2 minutes at 4℃ and 13,000 rpm. 9. Take the supernatant to the new tube, add chloroform + isoamyl alcohol 42 μL. 10. Vortex 1 minute, and Centrifuge for 2 minutes at 4℃ and 13,000 rpm. 11. Take the supernatant to the new tube, add 4.2 μL 3 M Sodium acetate, 42 μL isopropanol, on ice for 5 minutes. 12. Centrifuge for 10 minutes at 4℃ and 15,000 rpm. 13. Discard the supernatant, add 1 mL 75% EtOH to wash, discard EtOH. 14. Add 1 mL 75% EtOH, Centrifuge for 10 minutes at 4℃ and 13,000 rpm. 15. Discard the supernatant, in the biological safety cabinet (BSC) dry out RNA. 16. Add 104 μL DEPC-treated water to reconstitution RNA.   Before aminoacylation, sample pretreatment to refold tRNA correctly  tRNA reannealing   1. Dilute the sample two times. 2. Heat the solution at 95℃ for 2 min. 3. 20~22℃ for 3 min. 4. 37℃ for 5 min. 5. Put the samples on ice and cool down. 6. Collect the samples and store at -80℃.   Note: To prevent the sample from degrading, do not heat or cool the sample gradually. |

9. Aminoacylation

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| Materials  Equipment:   1. PCR tubes 2. Pipette and pipette tips 3. Ice bucket 4. Centrifuge   Consumables:   1. Purified tRNA 2. Amino acid 3. 1 M HEPES-KOH (pH 7.6) 4. 100 mM ATP (pH 7) 5. 100 mM DTT 6. 3 M KCl 7. 1 M MgCl2 8. Aminoacyl-tRNA synthetase 9. 3 M NaOAc (pH 5.2) 10. Phenol saturated with 300 mM NaOAc (pH 5.2) 11. CHCl3 and isoamyl alcohol (24:1 premix) 12. 75% ethanol 13. 100% ethanol 14. YM-10 column | Protocol   1. Mix: In a reaction volume of 500 μL, combine the following components: 120 ug tRNA, buffer [100 mM HEPES-KOH (pH 7.6), 20 mM MgCl2, 10 mM KCl, 1 mM DTT], 10 mM ATP, 100 uM amino acid, 1 mM AARS. 2. Incubate: Incubate the reaction in a circulating water bath at 37℃ for 20 minutes. 3. Extraction:    1. Add 50 μL of 3 M NaOAc (pH 5.2).    2. Extract with an equal volume of phenol for 10 minutes using a vortex-mixer in the cold room.    3. Separate the phases by spinning them in a microfuge for 5 minutes at 13000 rpm. Move the aqueous phase to a new microfuge tube.    4. Back extract the phenol phase with 300 mM NaOAc (pH 5.2), spin, remove the aqueous phase and combine it with the aqueous fraction saved from step (3).    5. Repeat step (3), (4).    6. Extract the aqueous phase twice with a mixture of CHCl3 and isoamyl alcohol (24:1 premix).- Precipitate:      1. Add 2.5 volumes of 100% ethanol to precipitate the aminoacyl-tRNA.      2. Centrifugal for 15 minutes at 4℃ and 13000 rpm, and remove supernatant.- Wash pellet: wash with 75% ethanol      - Reconstitution: Dissolve pellets in 20 μL of 2 mM NaOAc (pH 5.2).      - Pass the aminoacyl-tRNA through spin columns and centrifuge for 30 min at 4℃ and 10000 xg.      - Collect the solution above the membrane and store at -80℃. |

10. Conversion of seryl-tRNA to selenocysteinyl-tRNA

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| Materials  Equipment:   1. Incubator 2. Vacuum dryer 3. Thermocycler 4. PCR tubes 5. Pipette and pipette tips 6. Ice bucket 7. Centrifuge   Consumables:   1. 100 mM Hepes, pH 7.0 (2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid) 2. 10 mM MgCl2 3. 20 mM KCl 4. 1 mM dithiothreitol 5. 3 M potassium acetate (pH 4.6) 6. 75% ethanol 7. 100% ethanol 8. ddH2O 9. ATP 10. Sodium selenite 11. SelD 12. SelA | Protocol   1. Prepare buffer H: 100 mM Hepes (pH 7.0), 10 mM MgCI2, 20 mM KCl, 1 mM dithiothreitol 2. 70 pmol of seryl-tRNA were incubated for 30 minutes at 25℃ in 50 μL of buffer H with 22 pmol of purified enzyme (SelA), 70 pmol of purified SelD protein, 0.1 μmol of ATP, and 1 nmol of sodium selenite. (When adding the sodium selenite, it should be carried out in the hood.) 3. At the time intervals given, the reaction is stopped by the addition of 0.1 volume of 3 M potassium acetate and 2.5 volumes of 100% ethanol. 4. The sample was placed in ice for 40 minutes, and the precipitate formed was collected by centrifugation. (15 minutes at 10,000 rpm in an Eppendorf centrifuge) 5. Wash the pellet with 75% ethanol. 6. The pellet was dried *in vacuo*. 7. Add 4 μL DEPC-treated water to reconstitute the sample. |

11. Deacylation-Tris-HCl

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Incubator (37℃) 4. Centrifuge   Consumables:   1. 0.2 M Tris-HCl (pH 9.5) 2. YM-10 column | Protocol   1. Add 100 μL 0.2 M Tris-HCl. 2. Incubate in a circulating water bath at 37℃ for 60 minutes. 3. Transfer reaction product to YM-10 column, centrifuge for 20 minutes at 4℃ and 10000 xg. 4. Take the supernatant and store in -30℃ freezer. |

12. Deacylation-Nuclease S1

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Incubator (37℃) 4. Centrifuge   Consumables:   1. 5X Reaction buffer for S1 nuclease 2. S1 nuclease 3. Nuclease-Free Water | Protocol   1. Prepare the following reaction mixture:     |  |  |    | --- | --- |    | Components | Volume |    | tRNA | 30 μg (X μL) |    | 5X buffer | 18 μL |    | S1 nuclease | 3 μL |    | Nuclease-Free Water | to 90 μL (69-X μL) | 2. Incubate the mixture at room temperature for 30 minutes. 3. Stop the reaction by adding 2 μL of 0.5 M EDTA and heating at 70℃ for 10 minutes. 4. Transfer reaction product to YM-10 column, centrifuge for 20 minutes at 4℃ and 10000 xg. 5. Take the supernatant and store in -30℃ freezer. |

13. Urea PAGE

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Pyrex dish with dimensions larger than the gel 4. Glass plates 5. Spacers and 10-well comb (0.75 mm thick) 6. Casting stand and clamp 7. Electrophoresis rig 8. Power supply 9. Rotator   Consumables:   1. 40% acrylamide solution (mono:bis; 19:1 premix) 2. 3 M NaOAc (pH 5.2) 3. Urea 4. 10% ammonium persulfate (APS) 5. N,N,N',N'-tetramethylethylenediamine (TEMED) 6. Bromophenol blue 7. Methylene blue 8. Loading dye [100 mM NaOAc (pH 5.2), 7 M urea, 0.05% bromophenol blue] 9. Staining solution [500 mM NaOAc (pH 5.2), 0.06% methylene blue] | Protocol   1. Pour a 6.5% polyacrylamide gel containing 100 mM NaOAc (pH 5.2) and 8 M urea. To polymerize the gel, add 600 μL 10% APS and 60 μL TEMED per 100 mL of acrylamide solution. 2. Once polymerized, set up the gel. Use 100 mM NaOAc (pH 5.2) as a running buffer. 3. Add loading dye to the tRNA samples. Load approximately 40 pmol tRNA per lane. Do not heat samples before loading. 4. Electrophoresis for 40 minutes at 100 V, 300 A in the refrigerator. 5. Remove the spacers and then separate the plates. Carefully transfer the gel into ~ 500 mL of staining solution contained in a Pyrex dish. Place the dish on a rotator set at its lowest speed and allow the gel to stain thoroughly (~ 30 minutes). 6. Transfer the staining solution (which can be reused) into a large glass bottle using a funnel. This can be done by holding the gel against the bottom of the dish with one gloved hand while supporting the weight of the dish with the other. 7. Destain the gel with water. Several changes of water will be necessary for complete destaining. It works well to siphon the water from a corner of the dish. |

14. Gel Electrophoresis

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| Materials  Equipment:   1. Microwave 2. Electrophoresis comb 3. Electrophoresis trays 4. Pipette and pipette tips 5. Buffer tank 6. Voltage source 7. Glass bottle or Erlenmeyer flask   Consumables:   1. Agarose powder 2. TBE or TAE buffer 3. λ EH (λ DNA/EcoRI + HindIII) marker (2X) - Promega 4. 1X SLB or 10X SLB (sample loading buffer) 5. DNA sample 6. EtBr (Ethidium bromide) 7. RO (reverse osmosis) water | Protocol Agarose gel preparation:   1. Adjust the amount of agarose to get the desired gel concentration (in this protocol, we will be using 0.7% agarose). 2. Weight 0.7 grams of agarose powder and add it to a glass bottle or Erlenmeyer flask. 3. Measure 100mL of TBE buffer or TAE buffer and add it to the glass bottle or Erlenmeyer. 4. Put the mix into the microwave and heat it until the agarose is completely dissolved. 5. Let the mix cool down and pour it into the electrophoresis tray. 6. Set the electrophoresis comb and wait for the gel to solidify.   Sample preparation:  Samples are prepared in various ways depending on their purpose.   |  |  |  |  | | --- | --- | --- | --- | | Purpose | DNA solution | SLB | Total Volume | | Size checking | 2 μL | 10 μL (2X) | 12 μL | | Gel extraction | depends on how much DNA solution is extracted | ~5 μL (10X) | ~50 μL |   Agarose gel running:   1. When the agarose gel solidified, take out the electrophoresis comb. 2. Pour the TBE buffer or TAE buffer into the buffer tank. Note: Buffer used for running has to be the same as the buffer used to prepare the gel solution. 3. Put the gel in the buffer tank. 4. Load 5 μL λ EH (λ DNA/EcoRI + HindIII) marker (2X) in a well and load the DNA sample mix with SLB in the remaining wells. 5. Run for 20 minutes at 100 V. Note: Voltage and time vary.   Agarose gel visualization:   1. Take the gel out of the buffer tank. 2. Soak the gel in the EtBr solution for 8 minutes and in RO water for 30 seconds. 3. Take the gel out of RO water and visualize the DNA under a UV transilluminator. |

15. Gel Extraction and Purification

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| Materials  Equipment:   1. Eppendorf tubes 2. Collection tubes 3. Spin column 4. Pipette and pipette tips 5. Centrifuge 6. Scalpel 7. Dry Thermo Unit   Consumables:   1. Isopropanol 2. Binding buffer 3. Washing buffer 4. Elution buffer | Protocol  Gel excision, solubilization and DNA binding:   1. Excise band with scalpel and transfer to a new eppendorf tube. 2. Weigh the gel slice in a tube (by measuring the weight difference of an empty eppendorf and the eppendorf with gel slice in the tube). 3. Add 3 volumes of binding buffer to 1 volume of gel (per 100 mg gel add 300 μL binding buffer). 4. Incubate at 60℃ for 2-10 minutes (or until the gel has completely dissolved). 5. Add 1.5 volumes of isopropanol and invert the eppendorf 10 times. 6. Place a spin column in a provided collection tube. Transfer 700 μL sample to the spin column and centrifuge for 30 seconds at 12,000 rpm. 7. Discard flow-through and place the spin column back into the collection tube.   Wash:   1. Add 500 μL of washing buffer to the spin column and centrifuge for 30 seconds, 12,000 rpm. 2. Discard flow-through and place the spin column back into the collection tube. 3. Add 200 μL of washing buffer to the spin column and centrifuge for 5 minutes, 12,000 rpm. 4. Discard flow-through and place the spin column back into the collection tube.   DNA elution:   1. Transfer spin column to clean eppendorf. Add 50 μL of elution buffer to the spin column. Centrifuge for 2 minutes, 12,000 rpm. 2. Collect the pure sample in the eppendorf and discard the spin column.     Keep the DNA solution in the -20℃ freezer. |

16. Enzyme Purification

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| Materials  Equipment:   1. Sonication 2. Centrifuge 3. His-trap 4. Nanodrop 5. Pipette and pipette tips   Consumables:   1. 1 M imidazole 2. 5 M NaCl 3. 10 mM Tris-HCl buffer (pH 7.5) 4. 1 M Tris-HCl buffer (pH 7.5) 5. Binding buffer [10 mM imidazole, 0.5 M NaCl, 10 mM Tris-HCl (pH 7.5) buffer] 6. Elution buffer [300 mM imidazole, 0.5 M NaCl, 10 mM tris-HCl (pH 7.5) buffer] | Protocol   1. Suspend cells in 4 mL of binding buffer. 2. Disrupt the suspended cells with sonication. (Take a sample for 100 μL culture as cell suspension and mix with SDS-PAGE sample buffer.) 3. Centrifuge the disrupted cells for 10 minutes at 4℃ and 12000 rpm, then take the supernatant. (Take the sample for 100 μL culture as supernatant, and mix with SDS-PAGE sample buffer.) (Suspend the pellet with the binding buffer and take the sample for 100μL culture as pellet and mix with SDS-PAGE sample buffer.) 4. Apply to His-trap column which was washed with the binding buffer. 5. Wash the column with 5 mL of the binding buffer. 6. Elute the binding buffer protein by Elution buffer and fraction into 0.5 mL aliquots. 7. Measure protein amount by nanodrop and collect enzyme fractions. 8. Dialyze the fraction 3 times with 100 mL 10 mM Tris-HCl at 4℃. 9. Centrifuge the enzyme sample for 10 minutes at 4℃ and 12000 rpm, then transfer the supernatant to a fresh tube. 10. Determine the protein amount of the enzyme sample. (Take 1 μg and mix with SDS-PAGE sample buffer.) 11. Run SDS-PAGE. (cell suspension, supernatant, pellet, purified) |

17. SDS-PAGE

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| Materials  Equipment:   1. Eppendorf tubes 2. Pipette and pipette tips 3. Centrifuge 4. Vortex 5. Ice bucket 6. Empty box 7. Dry Thermo Unit 8. Glass plates 9. Spacers and 10-well comb (0.75 mm thick) 10. Casting stand and clamp 11. Electrophoresis rig 12. Power supply 13. Rotator   Consumables:   1. ddH2O 2. 30% acrylamide 3. 1.5 M Tris-HCl (pH 8.8) 4. 0.5 M Tris-HCl (pH 6.8) 5. 10% SDS 6. 10% APS 7. TEMED 8. Prestained Protein Marker - Bioman 9. Protein dye 10. Overnight culture (sample) 11. CBB (Coomassie Brilliant Blue) staining solution 12. Destaining buffer 13. 1X Tank buffer 14. Isopropanol | Protocol  Resolving gel preparation   1. In a 50 mL eppendorf tube add all the components 10% gel concentration.     |  |  |    | --- | --- |    | Components | Volume |    | ddH2O | 1.36 mL |    | 30% acrylamide mix | 1.675 mL |    | 1.5 M Tris-HCl (pH 8.8) | 1.875 mL |    | 10% SDS | 50 μL |    | 10% APS | 35 μL |    | TEMED | 4 μL |    | Total Volume | 5 mL |     (Adjust the amount added by this ratio) 2. Pour the mixture in between the glasses and add isopropanol afterward. 3. Wait for 10-15 minutes or until the gel solidifies. 4. Pour out the isopropanol.  |  |  | | --- | --- | | Components | Volume | | ddH2O | 2.1 mL | | 30% acrylamide mix | 500 μL || 0.5 M Tris-HCl (pH 6.8) | 375 μL | | 10% SDS | 30 μL | | 10% APS | 20 μL | | TEMED | 3 μL | | Total Volume | 3.028 mL |  (Adjust the amount added by this ratio)5. Pour the mixture in between the glass plates and add the 10-well comb. 6. Wait for 10-15 minutes or until the gel solidifies.   Sample preparation   1. Take overnight cultures with the desired volume and centrifuge. 2. Take 12 μL of pellet or supernatant (depends on necessity) and move to a new fresh eppendorf. 3. Add 3 μL dye into the eppendorf. Vortex and centrifuge briefly. 4. Heat the eppendorf at 100℃ for 10 minutes and centrifuge briefly. 5. Put samples in the ice bucket.   Gel running   1. When the gel solidified, set up the running equipment and pour 1X tank buffer. 2. Take out the comb and load 3 μL of the marker into a well and load the samples into the remaining wells. 3. Run the first 30 minutes at 120V then continue to run for 1 hour at 150 V. Note: voltage and time may vary.   Staining   1. Take out the glass plates out of the buffer tank and split up the glass plates to take out the gel. 2. The stacking gel and put the resolving gel inside an empty box, and then add the CBB staining solution until it covers the gel. 3. Put the box on a shaker and shake for 30 minutes or until the gel turns blue.   Destaining   1. Pour the CBB staining solution back to the bottle and add the destaining buffer into the box until it covers the gel. 2. Put the box on a shaker and shake until the protein bands are visible or until the gel turns white. 3. Pour out the destaining buffer. |

18. Electroporation *Escherichia coli* for homologous

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| Materials  Equipment:   1. Pipette and pipette tips 2. Eppendorf tubes 3. Centrifuge 4. Flasks 5. Electroporation cuvettes (2 mm gap) 6. Electroporator   Consumables:   1. SOB 2. Chilled 10% glycerol 3. SOC 4. Selection plates 5. DNA fragment 6. 10% L-arabinose 7. Ampicillin | Protocol  Competent cell making:   1. Inoculate 500 μL of overnight culture (*Escherichia coli* with pKD46) to 50 mL of fresh SOB-Mg medium with 50 μL of ampicillin at 30℃. 2. Grow cells with shaking at 30℃ for 1 hour and 50 minutes. 3. Add 1 mL of 10% L-arabinose and culture for 1 hour. 4. Transfer the culture into a 50 mL centrifuge tube, and centrifuge it for 5 minutes at 4℃ and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation.) 5. Remove the supernatant and suspend the cells with 1 mL of chilled 10% glycerol. Transfer the cell suspension into a 1.5 mL tube and centrifuge again for 1 minute at 4℃ and 12,000 rpm (first wash). 6. Remove the supernatant using a pipette and suspend the cells with 1 mL of chilled 10% glycerol. Centrifuge again for 1 minute at 4℃ and 12,000 rpm (second wash). 7. Remove the supernatant using a pipette and suspend the cells with 1 mL of chilled 10% glycerol. Centrifuge again for 1 minute at 4℃ and 12,000 rpm (third wash). 8. Remove the supernatant using a pipette and suspend the cells with 250 μL (depends on the amount of the cell) of chilled 10% glycerol.   Electroporation:   1. Mix 50 μL of the competent cell and 5 μL DNA solution in a fresh 1.5 mL tube. 2. Transfer the mixture into a chilled electroporation cuvette (2 mm gap), and pulse (Ec 2). 3. Add 1 mL of SOC with 20 μL 10% arabinose immediately after the pulse. 4. Incubate it for 1 hour at 37℃. 5. Plate 100 μL of the culture on a selection plate, and incubate at 30℃. |

19. *Escherichia coli* Electroporation with plasmid

|  |  |
| --- | --- |
| Materials  Equipment:   1. Pipette and pipette tips 2. Eppendorf tubes 3. Centrifuge 4. Flasks 5. Electroporation cuvettes (2 mm gap) 6. Eletroporator   Consumables:   1. SOB 2. Chilled 10% glycerol 3. SOC 4. Selection plates 5. DNA plasmid | Protocol  Competent cell making:   1. Inoculate 1 mL of overnight culture to 50 mL of fresh SOB-Mg medium. 2. Grow cells with shaking at 37℃ for 1.5 hours (*recA*+ strain) or 1.75 hours (*recA*- strain). 3. Chill the flask on ice for 5 minutes, transfer the culture into a 50 mL centrifuge tube, and centrifuge it for 5 minutes at 4℃ and 6,000 rpm to harvest cells. (After this stage, the cells should be kept cool throughout the cell preparation.) 4. Remove the supernatant and suspend the cells with 1 mL of chilled 10% glycerol. Transfer the cell suspension into a 1.5 mL tube and centrifuge again for 1 minute at 4℃ and 12,000 rpm. 5. Remove the supernatant using a pipette and suspend the cells with 1 mL of chilled 10% glycerol. Centrifuge again for 1 minute at 4℃ and 12,000 rpm. 6. Remove the supernatant using a pipette and suspend the cells with 250 μL (depending on the amount of the cell) of chilled 10% glycerol.   Electroporation:   1. Mix 50 μL of the competent cell and DNA solution (the amount depends on the DNA plasmid) in a fresh 1.5 mL tube. 2. Transfer the mixture into a chilled electroporation cuvette (2 mm gap), and pulse (Ec 2). 3. Add 1 mL of SOC immediately after the pulse. 4. Incubate it for 1 hour at 37℃. 5. Place the culture on a selection plate, and incubate. |

20. Plasmid Extraction

|  |  |
| --- | --- |
| Materials  Equipment:   1. Eppendorf tubes 2. Collection tubes 3. Spin column 4. Pipette and pipette tips 5. Centrifuge 6. Ice bucket   Consumables:   1. Suspension buffer 2. Lysis buffer 3. Binding buffer 4. Washing buffer I 5. Washing buffer II 6. Elution buffer 7. LB broth 8. Antibiotic as needed | Protocol   1. Prepare 2-5 mL overnight culture in LB medium with the antibiotic (depends on the plasmid to be extracted). 2. Collect cells from the overnight culture with a centrifuge for 30 seconds at 12,000 rpm, then discard the supernatant. 3. Add 250 μL of suspension buffer and suspend bacterial cells by vortex. 4. Add 250 μL of lysis buffer and mix gently by inverting the tube 10 times. 5. Add 350 μL of binding buffer and mix gently by inverting the tube 10 times. 6. Incubate on ice for 5 minutes. 7. Centrifuge at 4℃, 12,000 rpm for 10 minutes. 8. Place a spin column in a provided collection tube. Transfer 750 μL of supernatant to the spin column. 9. Centrifuge at room temperature, 12,000 rpm for 30 seconds and discard the flow-through. 10. Add 500 μL of washing buffer I to the spin column and centrifuge for 30 seconds. Discard the flow-through. |

21. DNA Purification

|  |  |
| --- | --- |
| Materials  Equipment:   1. Eppendorf tubes 2. Collection tubes 3. Spin column 4. Pipette and pipette tips 5. Centrifuge   Consumables:   1. Binding buffer 2. Washing buffer 3. Elution buffer 4. MQ or ddH2O | Protocol  DNA binding:   1. Add MQ or ddH2O to DNA solution until the volume reaches 100 μL. 2. Add 500 μL of binding buffer to the solution. 3. Transfer sample to the spin column and centrifuge for 30 seconds at 12,000 rpm. 4. Discard flow-through and place the spin column back into the collection tube.   Wash:   1. Add 500 μl of washing buffer to the spin column and centrifuge for 30 seconds at 12,000 rpm. 2. Discard the flow-through and place the spin column back into the collection tube. 3. Add 200 μl of washing buffer to the spin column and centrifuge for 5 minutes at 12,000 rpm. 4. Discard the flow-through and place the spin column back into the collection tube.   DNA elution:   1. Transfer spin column to clean eppendorf. Add 50 μL of elution buffer to the spin column. Centrifuge for 2 minutes at 12,000 rpm. 2. Collect the pure sample in the eppendorf and discard the spin column. 3. Keep the DNA solution in -20℃ freezer. |

![](https://static.igem.wiki/teams/4171/wiki/movetop1new.png)
{% endblock %}

{% extends "layoutwithcontents5.html" %}
{% block title %}{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
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![](https://static.igem.wiki/teams/4171/wiki/result-f.png)

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 RiboMAXTM 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 tRNASer, tRNASec, and tRNAUTuX.

    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).

![](https://static.igem.wiki/teams/4171/wiki/vitro-1.png)

Fig. 1. Confirmation of *PT7* with tRNASer ([BBa\_K4171014](http://parts.igem.org/Part%3ABBa_K4171014)), tRNASec ([BBa\_K4171015](http://parts.igem.org/Part%3ABBa_K4171015)), and tRNAUTuX ([BBa\_K4171016](http://parts.igem.org/Part%3ABBa_K4171016)) sequence construction by PCR
 M: Marker; Lane1: PT7-tRNASer (111 bp); Lane2: PT7-tRNASec (118 bp); Lane3: PT7-tRNAUTuX (114 bp)

    T7 RiboMAXTM 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page) (Fig. 2).

![](https://static.igem.wiki/teams/4171/wiki/vitro-2.png)

Fig. 2. Confirmation of tRNASer ([BBa\_K4171007](http://parts.igem.org/Part%3ABBa_K4171007)), tRNASec ([BBa\_K4171008](http://parts.igem.org/Part%3ABBa_K4171008)), and tRNAUTuX ([BBa\_K4171009](http://parts.igem.org/Part%3ABBa_K4171009)) *in vitro* synthesis
 M: Marker; Lane1: tRNASer (88 bp); Lane2: tRNASec (95 bp); Lane3: tRNAUTuX (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](http://parts.igem.org/Part%3ABBa_K4171001)) was used in seryl-tRNA production, while SelA ([BBa\_K4171002](http://parts.igem.org/Part%3ABBa_K4171002)) and SelD ([BBa\_K4171003](http://parts.igem.org/Part%3ABBa_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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page), and tRNA was made to bind with Ser through enzyme SerRS and buffer (see [Experiments](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) 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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) page), and the result of Urea PAGE is shown below (Fig. 3 and 4).

![](https://static.igem.wiki/teams/4171/wiki/urea-ser-sec.png)

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

![](https://static.igem.wiki/teams/4171/wiki/urea-utux.png)

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

     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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) 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 tRNASer and tRNAUTuX are similar to those of Sec standard, which proves that tRNA *in vitro* Sec synthesis is successful.

                (A)

![](https://static.igem.wiki/teams/4171/wiki/engineerfig-11-a-png.png)

                (B)

![](https://static.igem.wiki/teams/4171/wiki/engineerfig-11-b-png.png)

Fig. 5. Confirmation of Sec by HPLC (A) Sec synthesized by tRNASer; (B) Sec synthesized by tRNAUTuX

    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.

|  |  |  |  |
| --- | --- | --- | --- |
|  | tRNASer | tRNASec | tRNAUTuX |
| Aminoacylation | Success | Success | Success |
| seryl-tRNA to selenocysteinyl-tRNA | Success | Fail | Success |

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

*In Vivo* Pathway

I. Construction success

Achievements:

1. Verification of CysK and CysE\* function
2. Construction of pSU-PlacI-cysK-cysE\* ([BBa\_K4171012](http://parts.igem.org/Part%3ABBa_K4171012))
3. Construction of pPompA-ydeD ([BBa\_K4171013](http://parts.igem.org/Part%3ABBa_K4171013))

Process:

    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, PlacI-cysK and PlacI-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.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/4171/wiki/cysk-function-test.png) Fig. 6. Examination of CysK function by transforming PlacI-cysK ([BBa\_K4171010](http://parts.igem.org/Part%3ABBa_K4171010)) into E. coli △cysK mutant | ![](https://static.igem.wiki/teams/4171/wiki/cyse-function-test.png) Fig. 7. Examination of CysE\* function by transforming PlacI-cysE\* ([BBa\_K4171011](http://parts.igem.org/Part%3ABBa_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.

![](https://static.igem.wiki/teams/4171/wiki/placi-cysk-cyse.png)

Fig. 8. Confirmation of PlacI-cysK-cysE\* by colony PCR
 M: Marker; Lane 1: PlacI-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 PompA-ydeD into the pathway, the total production of Sec is expected to increase, and *E. coli* should grow better.

![](https://static.igem.wiki/teams/4171/wiki/pompa-yded.png)

Fig. 9. Confirmation of PompA-ydeD ([BBa\_K4171013](http://parts.igem.org/Part%3ABBa_K4171013)) by colony PCR
 M: Marker; Lane 1: PompA-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-PlacI-cysK-cysE\* and PompA-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).

![](https://static.igem.wiki/teams/4171/wiki/strain-selection.png)

Fig. 10. Strain selection by measuring growth curves

![](https://static.igem.wiki/teams/4171/wiki/color-of-selenium-in-tubes.png)

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.

![](https://static.igem.wiki/teams/4171/wiki/cfu-yded-for-wiki.png)

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) ![](https://static.igem.wiki/teams/4171/wiki/standard-cys.png) | (B) ![](https://static.igem.wiki/teams/4171/wiki/standard-ser.png) |

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

|  |  |
| --- | --- |
| (A) ![](https://static.igem.wiki/teams/4171/wiki/result14a.png) | (B) ![](https://static.igem.wiki/teams/4171/wiki/result14b.png) |
| (C) ![](https://static.igem.wiki/teams/4171/wiki/result14c.png) | (D) ![](https://static.igem.wiki/teams/4171/wiki/result14d.png) |

Fig. 14. Confirmation of results 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

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](http://parts.igem.org/Part%3ABBa_K4171001)) 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-Ptrc-melA in DH5α by SDS-PAGE, and the result is shown below.

![](https://static.igem.wiki/teams/4171/wiki/ki-mela15.png)

Fig. 15. Confirmation of pSB4KI-Ptrc-melA ([BBa\_K4171018](http://parts.igem.org/Part%3ABBa_K4171018)) by digestion
M: Marker; Lane 1: pSB4KI-Ptrc-melA (6707 bp); Lane 2: pSB4KI-Ptrc-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*.

![](https://static.igem.wiki/teams/4171/wiki/mela-sds-page.png)

Fig. 16. The SDS-PAGE result of pSB4KI-Ptrc-melA ([BBa\_K4171018](http://parts.igem.org/Part%3ABBa_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) ![](https://static.igem.wiki/teams/4171/wiki/17a.jpg) | (B) ![](https://static.igem.wiki/teams/4171/wiki/17b.png) |

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

![](https://static.igem.wiki/teams/4171/wiki/melanin-table-2.png)
![](https://static.igem.wiki/teams/4171/wiki/strain-and-medium18.png)

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) ![](https://static.igem.wiki/teams/4171/wiki/cu19a.png) | ![](https://static.igem.wiki/teams/4171/wiki/cu-table-19a.png) |
 (B) ![](https://static.igem.wiki/teams/4171/wiki/temp19b.png) ![](https://static.igem.wiki/teams/4171/wiki/temp-table19b.png) | |

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.

![](https://static.igem.wiki/teams/4171/wiki/tyrp20-1.png)

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D) page.

|  |  |
| --- | --- |
| (A) ![](https://static.igem.wiki/teams/4171/wiki/melanin-survival-fig-2.png) | (B) ![](https://static.igem.wiki/teams/4171/wiki/mela-survival-fig-1.png) |

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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) 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.

![](https://static.igem.wiki/teams/4171/wiki/gaba-dual-plasmids-22.png)

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.

![](https://static.igem.wiki/teams/4171/wiki/gaba-substrate-23.png)

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) ![](https://static.igem.wiki/teams/4171/wiki/sec-survival-4a.png) | (B) ![](https://static.igem.wiki/teams/4171/wiki/sec-survival-fig3.png) |

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

![](https://static.igem.wiki/teams/4171/wiki/gaba-secoli25-1.png)

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*.

References

[1] UniProt. www.uniprot.org. Accessed September 24, 2022. https://www.uniprot.org/uniprotkb/P16456/entry
[2] UniProt. www.uniprot.org. Accessed September 24, 2022. https://www.uniprot.org/uniprotkb/P0A821/entry
[3] Fu, X., Crnković, A., Sevostyanova, A., Söll, D. (2018). Designing seryl‐ tRNA synthetase for improved serylation of selenocysteine tRNA s. *FEBS Letters*, 592(22), 3759-3768. doi:10.1002/1873-3468.13271
[4] Walker, S. E., Fredrick, K. (2008). Preparation and evaluation of acylated tRNAs. *Methods*. 44(2):81-86. doi:10.1016/j.ymeth.2007.09.003
[5] Forchhammer, K., Böck, A. (1991). Selenocysteine synthase from *Escherichia coli*. Analysis of the reaction sequence. *Journal of Biological Chemistry*, 266(10), 6324-6328. doi:10.1016/s0021-9258(18)38121-3
[6] Köhrer, C., RajBhandary, U. L. (2008). The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases. *Methods*, 44(2), 129-138. doi:10.1016/j.ymeth.2007.10.006
[7] S1 Nuclease (100 U/µL). www.thermofisher.com. Accessed September 26, 2022. https://www.thermofisher.com/order/catalog/product/EN0321
[8] Tetteh, A. Y., Sun, K. H., Hung, C. Y., Kittur, F. S., Ibeanu, G. C., Williams, D., Xie, J. (2014). Transcriptional response of selenopolypeptide genes and selenocysteine biosynthesis machinery genes in *Escherichia coli* during selenite reduction. *International Journal of Microbiology*. 2014:1-11. doi:10.1155/2014/394835
[9] Wiriyathanawudhiwong, N., Ohtsu, I., Li, Z. D., Mori, H., Takagi, H. (2009). The outer membrane TolC is involved in cysteine tolerance and overproduction in *Escherichia coli*. *Applied microbiology and biotechnology*, 81(5), 903-913. doi:10.1007/s00253-008-1686-9
[10] Wang, Z., Tschirhart, T., Schultzhaus, Z., Kelly, E. E., Chen, A., Oh, E., Nag, O., Glaser, E. R., Kim, E., Lloyd P. F., Charles, P. T., Li, W., Leary, D., Compton, J., Phillips, D. A., Dhinojwala, A., Payne, G. F., Vora, G. J. (2020). Melanin produced by the fast-growing marine bacterium *Vibrio natriegens* through heterologous biosynthesis: characterization and application. *Applied and Environmental Microbiology.* 86(5):e02749-19. doi:10.1128/AEM.02749-19
[11] Cao, W., McCallum N. C., Ni QZ, Li, W., Boyce, H., Mao, H., Zhou X., Sun, H., Thompson, M. P., Battistella, C., Wasielewski, M. R., Dhinojwala, A., Shawkey, M. D., Burkart, M. D., Wang, Z., Gianneschi, N. C. (2020). Selenomelanin: An abiotic selenium analogue of pheomelanin. *Journal of the American Chemical Society.* 142(29):12802-12810. doi:10.1021/jacs.0c05573

![](https://static.igem.wiki/teams/4171/wiki/result.png)

![](https://static.igem.wiki/teams/4171/wiki/movetop1new.png)
[Sec *in vitro*](#page1)
[Sec *in vivo*](#page2)
[Melanin](#page3)
[GABA](#page4)
[Selenomelanin](#page5)
{% endblock %}