Difference between revisions of "Part:BBa K1608000"

Latest revision as of 02:25, 28 October 2020

SR/pSB1C3 (Fire Retardant BioBrick Part)

We identified a novel fire retardant protein, phosphorylated SR protein, by data mining with the criteria of high contents of phosphoserine and arginine (the highest nitrogen ratio among 20 amino acids). If you want to produce a functional one, you have to phosphorylate SR protein with SRPK1 (BBa_K1608001). For more detailed description, please go to our project [http://2015.igem.org/Team:Mingdao main page] in Mingdao iGEM 2015.

SR protein

Serine/arginine-rich splicing factor 1 (SRSF1) is a protein found on chromosome 17 in humans. SRSF1 is also known as alternative splicing factor 1 (ASF1), pre-mRNA-splicing factor SF2 (SF2), which is involved in alternative splicing, mRNA nuclear export and translation. It contains two functional domains. One in the N-terminus is RNA recognition motifs which interact with RNA and splicing factors. The other in the C-terminus is rich in serine and arginine residues (SR domain) and regulate the protein activity through phosphorylation by SR protein kinase.

Flame test

WOODEN HOUSE

COTTON

Protein expression

Procedure of SDS-PAGE & Western Blotting

We transferred the SR and SRPK genes into pGEX-2T and pET-29b, respectively. Then, E. coli BL21 was co-transformed with SR/pGEX-2T (with a GST tag) and SRPK/pET-29b (with a His tag). Protein lysates were collected after IPTG induction and subjected to SDS-PAGE and Western Blot analyses.

To analyze protein expression, we run SDS-PAGE and performed Coomassie blue staining and western blotting with the following standard procedure. ↓ recover E. coli BL21 from the stock at -80°C in 1ml of LB media supplemented with 50ug/ml Ampicillin and 25 ug/ml Kanamycin and culture O/N ↓ 100X dilution in 3ml of LB media with antibiotics and culture for around 2 hr ↓ Until OD600 between 0.6 to 1.0, IPTG was added at the concentration of 1mM and culture for another 4 hr for protein induction ↓ bacteria were collected by centrifuged and lysed in lysis buffer (lysates can stored at -20°C ↓ After centrifugation, lysates were collected and combined with sample buffer followed by boiling at 95°C for 10 min ↓ Now the samples were ready for running SDS-PAGE ↓ To run PAGE, transfer and western blotting are following the manufacturers’ protocol.

Groups for protein analysis:

• 1. pGEX-2T and pET-29b empty vectors (as a vector only control)
• 2. SR/pGEX-2T with pET-29b empty vector (as an unphosphorylated SR control
• 3. SRPK1/pET-29b with pGEX-2T empty vector (as a SRPK1 only control)
• 4. SR/pGEX-2T plus SRPK1/pET-29b (for phosphorylated SR protein expression)

Table 1. Antibodies and chromogen we used in the project.

Results

Figure 1. SDS-PAGE stained with Coomassie Blue

Figure 2. Western blot analysis with anti-His antibody

Figure 3. Western blot analysis with anti-GST antibody

In Figure 1, SPPK was expressed at the predicted 93 kDa (Lane 3) and SR protein was at 53 kD (Lane 2). In addition, the free form of GST (Lane 1 & 3) can also be detected at around 27 kDa

In Figure 2, SRPK protein fused with 6XHis tag was detected by anti-His antibody and visualized with BCIP/NBT chromogen at the predicted size of 93 kDa in Lanes 3 and 4.

In Figure 3, SR protein fused with GST tag was detected by anti-GST antibody and visualized with DAB chromogen at the predicted size of 53 kDa in Lanes 2 and 4. Moreover, in Lane 4, the shifted bands indicated the possibility of various forms of phosphorylated SR proteins by SRPK1 kinase. Free forms of GST can be detected at 27 kDa in Lanes 1 and 3.

Summary

SR protein and SRPK protein can be expressed in E. coli BL21 carrying the corresponding plasmids, proving the successful protein induction by IPTG and gene expression by pGEX-2T and pET-29b vectors. Furthermore, SR protein modified by SRPK kinase can be clearly observed in the shifted bands, probably demonstrating the phosphorylation process.

Contribution

Team: PuiChing_Macau 2020
Summary: We have compared the original SR without adhesion domains and our engineered SR with adhesion domains, by the fire retardancy test. As shown in figure 1, the protein with adhesion domain has a great improvement on its fire retardancy.