Part:BBa_K5427003
MaSp1
Background Information
(A3I)3-A14 is a synthetic spider silk protein (MaSp1, spidroin) designed by Ardnt et al. in 2022. It was selected because the protein was designed to be expressed in a prokaryotic system as natural spider silk is derived from eukaryotic cells. In previous work by Andersson et al. in 2017, they designed a chimeric spidroin (NT2RepCT) for use in scaling up production. NT2RepCT is a miniature spidroin that allows for large-scale production of spider silk fibers in prokaryotes, however, these fibers have properties that are inferior to natural spider silk. Specifically, these artificial fibers are about 15% of the natural fiber strength. Despite this, natural spider silk proteins are larger and have natural secretory mechanisms that do not exist in Escherichia coli which is why the use of a miniature spidroin is necessary for scaling up production. NT2RepCT was then improved upon (Ardnt et al. in 2022). The miniature spidroin designed by Arndt et al. in 2022 is ideal as it contains alanine residues that may be secreted to be artificially spun into fibers unlike natural spidroin proteins that contain different hydrophobic amino acids that cannot be secreted as easily. This synthetic spidroin is a modified version of NT2RepCT as it also contains isoleucine to assist in its strength in the spun fiber. The selected mini-spidroin, ((A3I)3-A14, forms fibers with a toughness that is comparable to the native dragline silk. Similar to NT2RepCT, it has the natural N and C termini from different spider species incorporated into its sequence.
Design Considerations
The MaSp1 sequence was optimized for E. coli expression by performing codon optimization and manually removing repetitive sequences to maintain genetic stability. We reviewed and adjusted the sequence to reduce complexity, repeating the process until complexity was minimized and no major issues remained. The GC content was manually adjusted (to 49.9%) to avoid high values, ensuring codons were spaced to prevent hairpin formations. Additionally, illegal restriction sites were removed to adhere to Biobrick standards. A His-tag was added to the N-terminus of the sequence based on the source information, facilitating easy protein purification. It is noted that in the future a plasmid containing tRNA for alanine and glycine would be necessary in order to optimize spider silk production, but we did not use that in our design.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Part Assembly
We tested the four previously constructed RBS-containing synthetic plasmid sequences, and the preliminary results indicated that RBS 1_12000 exhibited the highest growth potential for E. coli in LB media. Based on these findings, we selected RBS 1_12000 as the most suitable candidate for prokaryotic production of spider silk. Accordingly, we constructed the synthetic plasmid pJUMP24-pTac-RBS1-SpiderSilk-sfGFP using the following strategies. We linearized the vector pJUMP24-pTac-RBS1-sfGFP through PCR amplification to introduce Gibson assembly-compatible overlapping tail sequences, using forward and reverse primers #BBa_K5427034 and #BBa_K5427035, respectively. The forward primer was specifically designed to introduce a spacer between the sfGFP gene and the spider silk gene to provide the necessary structural flexibility. The spider silk gene block was ordered from IDT and amplified using primers #BBa_K5427036 (forward) and #BBa_K5427037 (reverse).
Characterization of MaSp1
We wanted to clone a Spider Silk producing gene into our previously tested plasmid (pTac_RBS1_Spider Silk_sfGFP_pJUMP 24) whose growth, chassis, and vector have undergone optimization. To test the effect that this Spider Silk gene has in our construct we performed another growth curve at 37C, using DH5alpha as our bacterial strain.
The results from our growth curve indicated that there was a significant difference between the two variables (empty vector versus construct). Each culture was grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm. The data showed that although both strains grew at a controlled rate, the empty vector seemed to outperform our construct. We saw that our construct variable’s exponential phase of growth lasted until around the 6 hour mark where we can see a beginning of plateauing into stationary phase. When we compared that with the empty vector growth, we saw a continuation of the stationary phase until the end of our experiment. Specifically at the 10 hour mark we observed a 48.03% reduction in growth of our vector in DH5a. We concluded that there must be some metabolic strain that our construct placed on the bacteria. Other literature has stated that in E. coli during silk protein synthesis upregulates stress response proteins, and therefore hinder growth significantly. Since we still observed growth, just significantly diminished, we determined that spider silk production would still be possible, but at a lower output than desired and that its growth rate does not affect biomass accumulation.
Figure 1 | Growth Curve for pTac_RBS 1_Spider silk_sfGFP_pJUMP 24 in E. coli strain DH5a and a DH5a empty vector control at 37 degrees grown in regular LB media. Each culture was grown for 10 hours and measurements of Optical Density (OD) at 600nm was taken every 2 hours. Each culture had 3 replicates grown and measured for OD, the average values were taken and plotted on the growth curve chart for analysis.
Protein Modeling
We modeled the monomer of the synthetic spider silk protein (mini-spidroin, (A3I)3-A14 to visualize the structure. The model of the monomer shows the formation of several alpha helices which is expected for soluble mini-spidroin according to Arndt et al. in 2022, specifically the N and C termini. There are regions of high and low confidence. The low confidence regions (alanine-rich) are involved in the protein-protein interactions when forming the spider silk fiber and should form beta sheets when the fiber forms. We also modeled the interaction of two monomers with AlphaFold2 in Neurosnap to visualize a potential interaction. This consisted of two identical chains of the mini-spidroin. This model also resulted in regions of high and low confidence. Spider silk fibers may consist of several proteins, therefore, it is likely that protein modeling consisting of several constituents of spider silk may be required to yield a high quality model. This may be a future avenue for iGEM teams to come. Furthermore, as suggested by Ardnt et al. in 2022, different amino acids may be ideal to form the beta sheets required in fiber formation which may be tested by future iGEM teams. The Ramachandran plots also highlight the regions with low pLDDT as these regions also had unacceptable phi and psi angles which suggests these regions are not accurately modeled with the current software available.
SpiderSilk Monomer Modeling
Figure 2 | Mini-spidroin (monomer) predicted local distance difference test (pLDDT) plot generated by AlphaFold2. Low pLDDT (confidence) regions represent the alanine-rich region of the protein involved in interactions that form the fiber.
Figure 3 | Mini-spidroin (monomer) multiple sequence alignment (MSA) sequence coverage plot generated by AlphaFold2.
Figure 4 | Mini-spidroin (monomer) model generated by AlphaFold2 and visualized with Jmol. GIF was generated by FirstGlance. Low confidence regions (red) represent the alanine-rich region of the protein involved in interactions that form the fiber.
Figure 5 | Mini-spidroin (monomer) Ramachandran plot generated by SWISS-MODEL.
SpiderSilk Dimer Modeling
Figure 6 | Mini-spidroin (dimer) predicted local distance difference test (pLDDT) plot generated by AlphaFold2.
Figure 7 | Mini-spidroin (dimer) multiple sequence alignment (MSA) sequence coverage plot generated by AlphaFold2.
Figure 8 | Mini-spidroin (dimer) model generated by AlphaFold2 and visualized with Jmol. GIF was generated by FirstGlance.
Figure 9 | Mini-spidroin (dimer) Ramachandran plot generated by SWISS-MODEL.
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