Data from Experimental Results

To access any information that is not directly available on this page, please check the documentation here.

Overview

The simulation confirmed the polymerization behavior of Actin as well as the interactions involving Thymosin β4 and Modified Thymosin. In the Experiment, we tested whether Actin polymerizes in real-world conditions and whether Thymosin β4 and Modified Thymosin could be successfully synthesized. 

Polymerize actin fiber

Objective

To check that Actin polymerization in real-world conditions follows the results obtained from the simulation. 

Method

We prepared three types of samples: one where Actin does not polymerize (Sample #1), one where Actin polymerizes (Sample #2), and one where Actin polymerizes and further bundles (Sample #3). For each sample, 5 μL was placed between a 40×22 mm and a 22×22 mm coverslip and observed using TIRF (Total Internal Reflection Fluorescence Microscopy).

The preparation recipes for the samples are shown in Tables 1 to 5 below.

Results

The observations confirmed using TIRF for Sample #1, Sample #2, and Sample #3 are shown in Figures 1 to 3. 

It was confirmed that Actin filaments were indeed formed. 

Synthesis of Thymosinβ4 and Modified Thymosin 

Overview

Thymosin β4 and Modified Thymosin were synthesized using the PURE system.

The general synthesis process is as follows:

A. 1st PCR

B. 2nd PCR

C. Preparation of templates for the PURE system

D. PURE system 

Since the amino acid sequences of Thymosin β4 and Designed Thymosin are known, reverse translation was performed to obtain their DNA sequences. 

The PURE system is a method for synthesizing proteins from DNA. However, the template DNA must include a T7 promoter sequence and a Ribosome Binding Site (SD sequence) upstream of the start codon of the target protein-coding gene.

To incorporate these sequences, PCR is performed twice to amplify the DNA. Using the DNA obtained for Thymosin β4 and Modified Thymosin, templates for the PURE system were prepared. The PURE system was then used to synthesize Thymosin β4 and Modified Thymosin.

The oligos used are listed in Table 6, with a total of seven types. 

Below, the method and detailed results of the synthesis experiment are described.

A. 1st PCR

PCR is performed to amplify the Thymosin β4 and Modified Thymosin sequences containing the SD sequence required for the PURE system.

Methods

First, the oligos were mixed and diluted.

Thymosin-oligo-f and Thymosin-oligo-r were mixed in equal amounts to prepare a 100 µM Thymosin template. Similarly, Design-Thymosin-oligo-f and Design-Thymosin-oligo-r were mixed in equal amounts to prepare a 100 µM Modified Thymosin template. The mixed oligo DNA (100 µM) was diluted 1000-fold with MilliQ water to a final concentration of 100 nM.

Next, the primers were diluted.

RBS-Thymosin-f (100 µM), Thymosin-r (100 µM), and Modified Thymosin-r (100 µM) were diluted to a final concentration of 4 µM using MilliQ water.

The first PCR was conducted. 

The PCR solution preparation is shown in detail in Tables 7 and 8 below.

The procedure of PCR is outlined in Table 9.

After PCR, electrophoresis was performed to confirm the presence of the desired DNA product.

The electrophoresis was conducted using PAGE with a SuperSep™ Ace, 10%, 17-well gel, and the buffer used was TakaRa TBE powder (#9121) dissolved in 1L of MilliQ water.

The contents loaded in each lane are shown in Table 10.

Electrophoresis was conducted at 135V for 45 minutes, followed by 200V for 20 minutes.

Results

The results of the electrophoresis are shown in Fig. 4

The correctly amplified product is expected to be 176 bp, and based on a comparision with the ladder in Fig. 4, it can be confirmed that DNA of approximately 176 bp was successfully amplified. 

B. 2nd PCR

Next, a PCR was conducted once more on the previously PCR-ed product to incorporate the T7 promoter sequence required for the PURE system and to amplify the product

Methods

First, the products from 1st PCR reaction #2 and #4 were column-purified to extract the DNA.

The detailed procedure for column purification is shown in Table 11.

The purified PCR product was diluted 100-fold by adding 99 µL of MilliQ water to 1 µL of the product.

Next, the second PCR was performed. The PCR settings remained the same as in the first round, as shown in Table 9.

The preparation of the solutions is detailed in Table 12 and Table 13.

After the PCR, electrophoresis was performed to verify whether the desired DNA was successfully amplified.

The electrophoresis was conducted using PAGE with a SuperSep™ Ace, 10%, 17-well gel, and the buffer used was TakaRa TBE powder (#9121) dissolved in 1L of MilliQ water.

The contents loaded into each lane are detailed in Table 14.

The electrophoresis was conducted at 135V for 45 minutes, followed by 200V for 20 minutes.

Results

The results of the electrophoresis are shown in Fig. 5.

The correctly amplified product is expected to be 234 bp, and based on the comparison with the ladder in Fig. 5, it is evident that DNA of approximately 234 bp was successfully amplified.

C. Template Preparation for PURE System 

The amplified DNA was extracted and then diluted for use in the PURE system.

Methods

The 2nd PCR reaction #2 and #4 were column-purified following the procedure outlined in Table 11, and the DNA was successfully extracted.

The concentration of the purified DNA was measured using the BioSpec Nano, and the concentration in ng/µL was converted to nM. Based on this calculation, a 20 nM PURE template was prepared.

Results

The results measured using the BioSpec Nano are shown in Fig. 6 and Fig. 7.

Fig. 6: Represents the concentration measurement of 2nd PCR reaction #2 (Thymosin β4).

Fig. 7: Represents the concentration measurement of 2nd PCR reaction #4 (Modified Thymosin).

The nucleic acid concentrations measured were as follows: Thymosin β4: 37.19 ng/μL and Modified Thymosin: 32.65 ng/μL

Using a band size of 234 bp, the concentrations were converted to nM. After that 20 nM PURE templates for both Thymosin β4 and Modified Thymosin were prepared by diluting the samples with MilliQ water.

D. PURE system 

Synthesize of Thymosin β4 and Modified Thymosin using the PURE system

Methods

Proteins were synthesized from DNA utilizing the PURE system. In this process, PUREfrex 2.0 was used. Details of the samples used in the reaction are shown in Tables 15 and 16.

The samples were incubated at 37℃ for 3 hours.

After the PURE system reaction, electrophoresis was performed to confirm whether Thymosin β4 and Modified Thymosin were synthesized. The analysis was conducted using Tricine-SDS-PAGE. The buffers used were as follows:

• Anode side: 0.1M Tris-HCl (pH 8.8)

• Cathode side: 0.05M Tris, 0.05M Tricine, and 0.1% SDS.

After running at 20A for 80 minutes, the procedure was continued at 40A for 12 minutes.

Results

The results of the electrophoresis are shown in Figure 8.

The molecular weight of Thymosin β4 was approximately 5 kDa, and comparing it with the ladder suggests that it was likely synthesized.

Conclusion

• It was confirmed that Actin polymerizes under real-world conditions.

• Thymosin β4 and Modified Thymosin were successfully synthesized from DNA. 

Author's Comment

Successfully synthesizing the newly designed protein is a giant step forward and will help us study how Actin behaves. Next, we want to do experiments to see how the Modified Thymosin and Actin interact with each other.

REFERENCE

[1]Shimizu, Y., Kanamori, T., & Ueda, T. (2005). Protein synthesis by pure translation systems. Methods, 36(3), 299-304. 

[2]Schägger, H. (2006). Tricine–sds-page. Nature protocols, 1(1), 16-22. 

[3]Promeg "FluoroTect™ GreenLys in vitro Translation Labeling System" https://ita.promega.com/-/media/files/resources/protocols/technical-bulletins/0/fluorotect-greenlys-in-vitro-translation-labeling-system-protocol.pdf?rev=f2ab366ddaad45cb8a23223f05756425&sc_lang=en (browsing 2024/11/15)

[4]Fuse-Murakami, T., Matsumoto, R., & Kanamori, T. (2024). N-Terminal Amino Acid Affects the Translation Efficiency at Lower Temperatures in a Reconstituted Protein Synthesis System. International Journal of Molecular Sciences, 25(10), 5264. 

[5]Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature biotechnology, 19(8), 751-755.