Difference between revisions of "Team:Mingdao/Project"

Credit: The Inquisitr News

Credit: fire-security.net

What’s fire? Fire is an oxidation-reduction chemical reaction. Combustion occurs between oxygen and fuel with heat over ignition temperature. The three elements are known as fire triangle. Free radicals formed in the burning process could spread the fire. Fire extinguished if one, or more, of four of them are removed or isolated.

In addition to fire retardant chemicals, there’s some natural products have been recognized as fire retardant bio-materials.

Credit: Ind. Eng. Chem. Res., 2014, 53 (10), pp 3917–3923

Credit: Freepik.com

Our goal of iGEM project this year is to find a novel fire retardant bio-material in addition to casein and wool. Furthermore, we’d like to make a fire retaradnt bacterium and produce fire retardant proteins. Finally, the prototype we create could be qualified through flame test and applied to the industry.

In order to find fire retardant protein candidates, we collected protein sequence data from Uniprot database and ranked with highest contents of serine (phosphoserine) and/or arginine (the highest nitrigen percentage among 20 amino acids). We chose a possible candiate (SR protein) in the top list of highest contents of both serine and arginine. Next, we built biobricks (pSB1C3) carrying SR gene and SRPK kinase gene which encodes proteins to phosphorylate SR protein, followed by putting the genes on the exprssion vectors of pGEX-2T and pET-29b. Then, we transformed and cultured bacteria (E. coli BL21) carrying the expression vectors. Finally, we analyzed the protein expression and performed flame test on cloth and cotton soaked with the bacteria.

Flow chart of finding and testing novel proteins with fire retardant properties

As mentioned earlier, phosphorous and nitrogen play important roles to stop fire propagation. We analyzed the contents of phosphorus and nitrogen among among amino acids (AAs).

Phosphorus exists as the form of phosphate. Phosphate group was only added on the AA by phosphorylation through a post-translational modification (PTM). Among all 20 AAs, only serine, threonine and tyrosine could be phosphorylated. Phosphoserine is the most common modification in phosphorylated proteins. Serine was chosen as the major source of phosphorus in proteins.

Nitrogen content analysis showed that arginine contains the highest nitrogen rate of 33% by weight among AAs. (American Journal of Plant Sciences, 2011, 2, 287-296)

Thus, we chose it as the major source of nitrogen in proteins.

Nitrogen contents in amino acids. Credit: American Journal of Plant Sciences, 2011, 2, 287-296

In order to find protein candidates with fire retardant properties, C programming was written for data mining. (For collaboration with NCTU-Formosa and the code programming data, see here)

We collected protein sequences from UniProt and downloaded data as fasta format on Feburary 13, 2015.

In the process, the sequence of a protein was read and the amount of each AA was added up, after that the AA amount was calculated into the rate of each AA by “ AA rate = (each AA amount)/(total AA in a protein)” by the C program automatically.

Next, the list of the AA rates of each protein was ranked by the (1) serine rate, (2) arginine rate ,and (3) serine plus arginine rate.

Finally, a manual curation was performed to remove the unknown, predicted or similar proteins.

The results of top 15 list of serine rate and arginine rate were shown below.

1.Top serine % of proteins in Uniprot database

Protein TPRXL is the highest serine rate by 67% and Protamine-B is the highest arginine rate by 70%. According to the unique roles and significant fire retardant effects played by phosphorus (serien) and nitrogen (arginine), serine plus arginine rate was calculated for our needs.

Top 10 of serine plus arginine rates of proteins in Uniprot was shown below.

Top 10 list of serine plus arginine rate showed that Protamine P3 and TPI6 are the top 2 proteins which are rich in arginine but short of serine. Serine/arginine-rich splicing factor 6 is more ideal for our criteria, which contains 33% of arginine and 40% of serine, matching our goal of finding a protein both rich in serine and arginine.

Serine/arginine-rich splicing factor 6 is a member belonging to SR protein family, which is a group of proteins conserved in serine/arginine-rich domain.

In our following experiments, we decided to use proteins in SR family as our target for bio-fire-retardant material.

SR protein family

Reference: Genome Biol. 2009, Biochem J. 2009

The SR protein family comprises a group of structurally related proteins with a conserved domain rich in serine (S) and arginine (R), which is so-called SR domain. They are involved in various mRNA metabolism and play essential roles in diverse cell functions such as constitutive and alternative pre-mRNA splicing, mRNA nuclear export, nonsense-mediated decay, and mRNA translation, etc. SR proteins are important in regulation of gene expression and developmental processes. SR domains are characterized by their ability to interact with other proteins and involved in nuclear translocation.

Which SR protein should we take?

The high serine and arginine contents of protein we predicted by data mining is Serine/arginine-rich splicing factor 6 from the species of Oryctolagus cuniculus (Rabbit). We conducted research on the Internet and take a trip to Academia Sinica, Taiwan to meet expert in SR protein research, Dr. Woan-Yuh Tarn. She did a lot of researches on SR protein and now is transfering interests to mRNA metabolism. She suggested Serine/arginine-rich splicing factor 1 in human with a long stretch of SR domain. And she has materials of SR gene for gene cloning and protein expression as well as SRPK1 gene encoding SR protein kinase, which can phosphorylate SR protein.

Serine/arginine-rich splicing factor 1

Reference: Wikipedia

Serine/arginine-rich splicing factor 1 (SRSF1) is a protein in human encoded by SFRS1 gene, which is located on chromosome 17. SRSF1 is essential for alternative splicing, mRNA nuclear export and translation. The function of SRSF1 is regulated by phosphorylation at the serine residues in SR domain by SR specific protein kinase 1 (SRPK1). And up to 12 phosphoserine sites have been charactized (J Mol Biol. 2008) The phosphorylated SR protein plays crucial roles in heart development, embryogenesis, tissue formation, cell momtility, etc.

SRSF1 amino acid sequence with SR domain

MSGGGVIRGPAGNNDCRIYVGNLPPDIRTKDIEDVFYKYGAIRDIDLKNRRGGPPFAFVEFEDPRDAEDAVYGRDGYDYDGYRLRVEFPRSGRGTGRGGGGGGGGGAPRGRYGPPSRRSENRVVVSGLPPSGSWQDLKDHMREAGDVCYADVYRDGTGVVEFVRKEDMTYAVRKLDNTKFRSHEGETAYIRVKVDGPRSPSYGRSRSRSRSRSRSRSRSNSRSRSYSPRRSRGSPRYSPRHSRSRSRT*

SRSF1 has 248 amino acids with a long stretch of SR domain in the C terminus containing a total of 43 arginine (17.3%) and 28 serine (11.2%). The comparison of protein composition between SR protein, wool and casein were made in the following table.

Comparison of phosphorus and nitrogen contents in fire retardant protein candidates, SRSF1, wool and casein

*Nitrogen content calculation : American Journal of Plant Sciences, 2011, 2, 287-296

**Phosphoserine : phosphorylated serine residues

SRSF1 has more nitrogen contents than wool and casein. The serine contents and phosphoserine residues characterized in SRSF1 are also greater than casein. Based on the analysis, the novel fire retarant protein, SRSF1, theoretically, could have better effect than wool and casein.

We got the plasmids of SRSF1 gene encoding SR protein and SRPK1 gene encoding protein kinase to phosphorylate SR protein from the lab of Dr. Woan-Yuh Tarn.

First of all, we cloned SR gene and SRPK1 gene onto the standard BioBrick, pSB1C3. Then, SR and SRPK1 genes were transferred to the commercial expression vectors of pGEX-2T and pET-29b, respectively, which have strong expression ability through IPTG induction process.

All the constructs have been confirmed by colony PCR, restriction enzyme check and sequencing. (For more detail, please see here)

We transformed E. coli BL21 with the following groups of vectors and made stocks at -80°C:

1.SR/pGEX-2T plus SRPK1/pET-29b (for phosphorylated SR protein expression)

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.pGEX-2T and pET-29b empty vectors (as a vector only control)

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.

Antibodies we used to blot the protein and predicted molecular weight of indicated proteins are described below.

In 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.

Figure 1.

Burning lenghts of cloth samples soaked with E. coli BL21 expressing proteins.

Figure 2.

Burning lenghts of cotton samples soaked with E. coli BL21 expressing proteins.

Taken together, the data of flame test strongly proved that phosphorylated SR protein is able to retard the fire propagation and further extinguish the burning. Moreover, the unphosphorylated SR protein has also some effect on fire retardancy. The results are not only demonstrating phosphorylated SR protein is a novel fire retardant proteins, but also verifying our theoretical hypothesis of phosphoserine and nitrogen contents that play unique and additive roles on the fire retardant properties.

Casein and wool are natural products with fire retardant properties. Phosphorus and nitrogen play important roles on the fire retardancy. We searched the highest (phospho)serine and highest arginine (nitrogen) contents in the protein databank of Uniprot by data mining. SR proteins are a family of proteins conversed in serine/arginine-rich doman and highly be phosphorylated. We expressed SR protein and SRPK1 kinase protein in E. coli BL21, which were analyzed in SDS-PAGE with coomassie blue staining and Western blotting with specific antibodies. Next, flame tests were performed on cloth and cotton samples soaked in the culture media containing protein-producing bacteria, demonstrating the fire retardant efficacy of phosphorylated SR proteins.

In protein analysis of phosphorylated SR proteins (section “Protein Expression”, Figure 3), total lysates were blotted with anti-GST antibody. The data showed various levels of band shift on SR plus SRPK (Lane 4) compared to SR only (Lane 2) around 53 kDa, indicating modification of SR protein. This result highly suggests SR protein underwent phosphorylation due to the presence of SRPK kinase. On the other hand, we’er trying to blot the lysates of phosphorylated SR protein with anti-phosphoserine antibody. However, we technically failed the experiment  and didn’t get a good result with anti-phosphoserine antibody.

Phosphorus and nitrogen are important on the effect of fire retardancy. We tried to synthesize a gene encoding a polypeptide with arginine repeat. However, IDT company, a sponsor of iGEM, replied us a technical difficulty on synthesizing nucleotides with high frequency repeats. As for phosphorus, as we mentioned, proteins don’t carrying any phosphate groups until being phosphorylated by specific kinase. Therefore, finding an existing protein and using an identified kinase are ideal to make phosphorylated proteins.

Currently, we don’t compare SR proteins to casein or wool on fire retardant efficiency. Because the cost and time to purify SR proteins are still problems for us so far. We are looking for companies who might have interests in fire retardant bio-materials sponsoring us to solidify our discovery.

In summary, our work is a proof-of-concept study of finding and verifying a novel fire retardant natural protein candidate of SR protein through data mining and flame test. Casein and wool have been proved as natural, non-toxic fire retardant bio-materials. And both of them have been patented. The SR protein we found could be the next-generation biomaterial for studying fire retardant effectiveness.