Latest revision as of 07:03, 11 October 2023

PySp1

PySp1 is the DNA sequence of a repetitive region of the piriform glandular filament of Arachnid. The function of the repeat area is mainly related to the mechanical properties of spider silk fiber.

Sequence and Features

2023 OTIA-Hangzhou’s Characterization of BBa_ K4413007

This year, our team has made a new characterization of the existing part, R (BBa_K4413007, a repetitive region in pyriform silk gene PySp1), for Bioplus-China 2022, which contributed some of meaningful data and conclusion. The addition of new data on the protein expression of R (BBa_K4413007) is expected to be helpful for future iGEM teams.

Experience and results

Prokaryotic expression of large amounts R proteins is very important for the next experiment. We tried to increase the expression of R protein by changing the induced expression temperature of R protein.

Constructed the R_pET21-a (+) plasmid

The prokaryotic expression system of spider silk (R) protein was constructed. pET-21a (+) was used as the recombinant protein expression plasmid backbone, and the plasmid construction map is shown in Fig. 1. This series of vectors is the most commonly used prokaryotic expression system, and the expression of target proteins is controlled by lactose manipulation elements.

Fig. 1 The recombinant plasmid of PySp1(R)_pET21-a (+)

The prokaryotic expression system of protein R was constructed by double enzyme digestion. First, the R was amplified from the plasmid which has the original sequence of spider silk gene using primers with enzyme cleavage sites, as shown in Fig. 2, and the purified PCR product (675 bp) was connected to the pET-21a (+) vector.

Fig. 2: The target gene R was amplified by PCR M: DNA marker; 1-4: The target gene R.

The correctly sequenced recombinant plasmid was transformed into the expressing strain E. coli BL21 (Fig. 3). The colony PCR shows that the PCR product was about 700 bp (Fig. 4), then selected the right colony for the next experiment.

Fig. 3 A plate of growing R_pET21-a (+) colonies

Fig. 4: Colony PCR of R_pET21-a (+) M: DNA marker; 2-5: The PCR products was about 700 bp.

Protein expression

2 mL of overnight cultured bacterial LB solution was added to 50 mL of LB liquid medium containing ampicillin antibiotics. And cultured at 37℃ at 200 rpm on a shaking incubator to OD600 of about 0.5. Then final concentration 0.5 mM of IPTG was added and cultured at 15℃ and 37℃. As shown in Figure 5, protein expression was observed after culture at 15℃ for 16 h in a 200 rpm shaker. Fig. 6 shows culture at 37 ° C for 16 h in a 200 rpm shaker. The SDS-PAGE shows that R protein mainly exists in the form of inclusion bodies, and the expression of R protein cultured at 37℃ is more than that at 15℃.

Fig. 5: The induced expression of protein R at 15℃ M: Protein marker; 1: Before IPTG induction; 2: The supernatant from ultrasonication after IPTG induction; 3: The precipitate from ultrasonication after IPTG induction.

Fig. 6: The induced expression of protein R at 37℃ M: Protein marke; 1: Before IPTG induction; 2: After IPTG induction; 3: The supernatant from ultrasonication after IPTG induction; 4: The precipitate from ultrasonication after IPTG induction.

Other research on optimized expression and enhanced production of spider silk protein

Inmaculada Jorge et al. used genomics and proteomics methods to study the differences in spider silk protein expression in different spiders, and found a variety of different gene translation patterns, which provided in-depth insights for further study of different spider silk protein expression [1].

Gargi Bhattacharyya et al. artificially increased expression levels of recombinant spider silks that are rich in glycine and/or proline in Nephila clavipes, the p4GPP plasmid was generated by modifying the pACYC184 vector (New England BioLabs) where glyT, proL, proM and glyVXY gene cassettes from E. coli were introduced in the pACYC184 vector to produce additional tRNAs for glycine and proline. In addition, spider silk-like genes with codon usage optimized have been designed for E. coli to reduce the chance of translation pauses through the formation of mRNA secondary structures and optimized protein translation by supplying a tRNA pool required for the Gly-Ala-Pro-rich mRNA transcripts of the silk-like genes [2].

Efficient and low-cost acquisition of spider silk protein is a challenge in this field, and we believe that this goal can be achieved through synthetic biology methods.

Reference:

[1] Jorge Inmaculada, Ruiz Víctor, Lavado-García Jesús et al. Expression of spidroin proteins in the silk glands of golden orb-weaver spiders[J]. J Exp Zool B Mol Dev Evol, 2022, 338: 241-253.

[2] Bhattacharyya Gargi, Oliveira Paula, Krishnaji Sreevidhya T et al. Large scale production of synthetic spider silk proteins in Escherichia coli[J]. Protein Expr Purif, 2021, 183: 105839.