Engineering Success

Cycle 1

Design

The primary structure of PySp1 (R in brief) includes repeated sequences and N-terminal and C-terminal. The function of N-terminal and C-terminal is to promote the assembly of spider silk protein to form spider silk fiber and the function of the repeat region is mainly related to the mechanical properties of spider silk fiber.

We planned to design a complete pathway for expression of 1- 3 repeats in Escherichia coli, and therefore, we first constructed an expression plasmid containing a repeat sequence, and expressed it in E.coli (BL21 strain), and then explored its physicochemical characteristics through subsequent characterization experiments.

Build

We obtained the repeat region sequence of protein PySp1 from the literature, and named it R in brief, and designed a prokaryotic expression system to express it. The composition part consist of T7 Promoter (BBa_I719005)、RBS (BBa_B0034)、R(BBa_K4413002)、T7 Terminator(BBa_K731721) (Figure 1). The T7 promoter controls the start of transcription, RBS sequence combines with ribosome to initiate the protein expression, and the 6X His sequence were inserted before the R for further protein purification. The target fragment was synthesized by a biological company, and then we recombined the R gene into plasmid with BamHⅠ and EcoRⅠ (Figure 2). In the end, we have successfully got the pET21a-R plasmid (Figure 3), and transformed it into E.coli (BL21 strain).

Test

We verified the length of R and DNA sequence through PCR (Figure 4) and sequence alignment (Figure 5), The following results can prove that we have successfully constructed the prokaryotic expression system of R.

The R expression in E. coli was induced, and the bacterial solution was preliminarily verified by SDS-PAGE (Figure 6). After that, the target bands in the gel were cut off for mass spectrometry analysis (Figure 7).

The LC-MS analysis indicates that 13 unique peptides cover >50% amino acid sequence of Protein R can be detected by Thermo Q-exactive MS. This result is also further proof that the target band is indeed the Protein R. Finally, we purified the Protein R from the inclusion body with urea.

Learn

After extensive experimental validation, we constructed recombinant plasmids and expressed them in E.coli. Finally we purified the Protein R from the inclusion body with urea, and mixed them with sodium alginate solution to form a composite hydrogel as a bioink feedstock for subsequent 3D printing. At the same time, in the Human Practice part, we realized the importance of intuitively observing the distribution of Protein R in hydrogels. Furthermore, both the tracking and maintenance of the bioink need a reliable indicator. Therefore, we decided to insert a fluorescent tag before the R gene to tracing the Protein R in our system.

Cycle 2

Design

BBa_K4413008 contains RBS and R genes. We plan to improve this cassette, and constructed the improve part (BBa_K4413009). We inserted an EGFP sequence in the upstream of R. The composition part consist of pT7(BBa_I719005), RBS(BBa_B0034)、EGFP(BBa_S03452), and R（BBa_K4413002）（Figure 1)。

Build

We designed a prokaryotic expression system with fluorescent tags, inserting the EGFP sequence upstream of the R to make the protein show green fluorescence. The EGFP fragment is cloned from the plasmid by PCR, and the 5'end of the primer used in PCR contain the upstream and downstream homology arms of the pET21a gene respectively，the PCR fragment size is 1395 bp (Figure 2). Integration of EGFP fragments and linearized vectors pET21a through homologous recombination (Figure 3), Finally, we successfully recombined the pET21a-EGFP-R plasmid (Figure 4), and transferred it into E. coli (BL21) strain.

We verified the length of EGFP-R sequence by PCR (Figure 5), and induced the BL21 strain by IPTG , Then we can observed obvious green fluorescence with fluorescence microscope.

Test

EGFP is an excellent reporter gene, which can mark and locate the fusion protein. Through fluorescence microscope, we can accurately locate the expression position of protein R (Figure 6), and we mix the purified protein with sodium alginate hydrogel (Figure 7), making our product more intuitive and convenient for positioning in subsequent experiments.

Learn

We have successfully constructed the prokaryotic expression vector of EGFP-R fusion protein. Compared with the original part, the fusion protein can be directly observed by fluorescence microscope or even eyes. Thus, we can now both increase the mechanic properties of the hydrogel, and locate our printed biomaterials for further tracing and maintenance.

Reference

Blasingame E, Tuton-Blasingame T, Larkin L, Falick AM, Zhao L, Fong J, Vaidyanathan V, Visperas A, Geurts P, Hu X, La Mattina C, Vierra C. Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus. J Biol Chem. 2009 Oct 16;284(42):29097-108. doi: 10.1074/jbc.M109.021378. Epub 2009 Aug 7. PMID: 19666476; PMCID: PMC2781455.