Team:HKUST/Experiments
Wet Lab
Basic Lab Plans
Sadly, due to the outbreak of COVID-19, access to the laboratory was restricted. Still, we made sure to plan out our lab work in detail. The following is a brief overview of our plans.
In this phase, our lab works were centered at:
- The overexpression of recombinant hagfish slime intermediate filament(I) subunits
- Fusion of chromoprotein on IF subunits for color mixing
- Draw-processing for properties enhancement and measurement of thread mechanics
Cloning
All clones utilized the high copy-number pSB1C3 backbone as transformation vectors to carry the two hagfish slime intermediate filament (IF) subunits, 6x his-tag, secretion peptide, and chromoprotein genes. Most DNA fragments are synthesized by IDT while some chromoprotein sequences were ordered from Twist Bioscience. For detailed information on the BioBrick we used and all clones we constructed as mentioned follows, click Parts to see.
All fragments are geared with a standard prefix, with EcoRI, Xbal restriction site, and a standard suffix, with SpeI, PstI restriction site. Chromoproteins genes are additionally flanked by Acc65I and AscI restriction sites for its insertion upstream or downstream of the IF subunits sequence respectively.
The first objective, boosting expression efficiency of the subunits, is done by testing different strong promoter sets, and by inserting a secretion peptide after our gene of interest. There are two promoter-RBS sets, K88 (BBa_K880005) and T7 (BBa_I712074). We hope to identify and use the one with higher translational efficiency for subsequent experiments. Another approach is, by tagging the subunits with a short secretion peptide, TorA (BBa_K3114005) or HlyA (BBa_K554002), we hope to enhance protein expression yield by mitigating inclusion body formation. (Sørensen and Mortensen, 2005).
Fusion of chromoprotein for coloration of the thread is another focus of our project. After identifying the most efficient construct, chromoproteins of different CMY colors (blue/red/yellow) are being linked to the subunits through the Acc65I restriction site at the N-terminal, or AscI restriction site at the C-terminal.
Each recombinant plasmid is then transformed into E. coli. (DH5α strain). The cells were made competent with the inoue method. The transformed colonies are cultivated, and colony PCR is performed. Successfully recombinant plasmids are purified with the Thermo Scientific GeneJET Plasmid Miniprep Kit for subsequent use.
Expression and Purification
Same E. coli. Strand (DH5α) as mentioned in the gene cloning part is used for protein expression. After transforming the competent cells with the desired recombinant plasmids, pre-culture containing: LB, chloramphenicol and a picked single colony is incubated overnight at 37 °C with vigorous shaking.
2ml of the pre-culture is then transferred into a larger container with fresh chloramphenicol-containing LB medium, and incubated under the same condition mentioned above, until OD600 reached 0.6-0.8. IPTG is then added to stimulate protein expression. The culture is further incubated for 3 hours.
For construct with no secretion peptide, the subunits produced are likely to aggregate in cells and form insoluble inclusion bodies. The purification process has to be done under denaturing conditions. Cells are harvested by centrifugation and lysed by vortexing in buffering solution with 8M urea as a denaturing agent. The clear supernatant is saved after centrifugation for further purification.
For construct with TorA secretion peptide inserted, the subunits will be expressed to the periplasm. Cells are harvested by centrifugation and resuspended in ice-cold magnesium sulphate. It serves as an osmotic shock agent which leads to the release of periplasmic proteins. The solution is then centrifuged and the supernatant is retained.
For construct with HlyA signal peptide, the tagged subunits will be expressed to the extracellular space. The supernatant will be used for purification after centrifugation.
Protein sizes and abundance are evaluated by SDS-PAGE before performing Ni-NTA column chromatography for the 6x His-tagged subunits. The yield of subunits is further analyzed using NanoDrop Microvolume Spectrophotometers and compared with different constructs.
The purified proteins are then cleaved with TEV protease to remove to 6x His-tag residues. Pure IF subunits can be obtained after running a size exclusion chromatography to exclude the his-tag residue and TEV protease in the solution. The resulting pure protein
As you might notice, the current purification method is tedious and too expensive to be applied in an industrial setting for mass production. Therefore, a novel purification method that does not rely on ordinary laboratory chromatography techniques is proposed. For more, please see project design and future plans.
IF Assembly and thread formation
The two purified subunits (alpha and gamma) are then mixed in a 1:1(w/w) ratio to undergo stepwise urea dialysis from 8 M to 0 M in Tris buffer of pH 8.4 at 4°C for self assembly of coil-coiled structure [1]. The resulting mixture can be freeze-dried and stored at 4°C for later use.
The freeze-dried mixture is then re-dissolved in 98% formic acid at 10% (w/v) concentration. Diluted with fresh formic acid, protein dope solutions prepared at different concentrations. Appropriate concentration of proteins dope solution is critical for the efficient production of mechanically well-performed threads. Increasing concentration of the solution manifests a positive relationship with thread diameter but is negatively correlated with threads of mechanical properties [2]. Dope solution concentration ranging from 5% to 10% will be used. Suitable amounts of the dope solution should be prepared upon demand to prevent degradation of IF in the doping solvent.
The simplest method to fabricate recombinant hagfish slime thread is to apply the protein dope solution on a surface of ice-cold magnesium chloride buffer and use a tweezer to slowly pick up the coagulated film from the solution interphase. The film will collapse due to gravity and forming a thread-like structure. The thread is then placed on a ventilated surface for overnight air dry.
More high-tech spinning methods, such as wet-spinning, electrospinning, and microfluidic devices can be investigated to enhance the efficiency of hagfish slime IF for mass production.
Colour Mixing
In this phase, the colouration of threads is focused on the fusion of chromoproteins. As mentioned in the cloning section, chromoprotein is being inserted into the circuit after the most efficient construct is identified. The interference of thread properties by directly linking a chromoprotein is first being tested. After confirming the feasibility (i.e. the fusion protein can still form a proper thread), colour mixing by assembling subunits with different colours will begin. In order to identify the correct ratio of each colour and to better project the final colour of the resulting thread, the subtractive colour mixing model was worked in the dry lab.
In case if the linkage of chromoprotein greatly deteriorates the quality of the thread, or leads to self-assembly failure. Chromoproteins will be directly expressed and purified. By mixing different ratios of chromoprotein, solutions of different colours can be obtained. Hagfish slime thread is then directly immersed into the chromoprotein mixture for colouration. This method will be performed in parallel with dyeing the thread with natural dye, such as indigo. Other methods of non-covalently linking chromoproteins to IF will be explored in phase 2.
Draw-processing and mechanical enhancement
It comes to the final step of the wet lab work, which is to artificially transform the alpha helix coiled-coil structure of hagfish slime intermediate filament to cross beta-sheet and eventually aligned beta-sheet. The molecular modelling of the transition is shown in the dry lab page The process is necessary for the thread to manifest outstanding mechanical properties that are comparable to spider silk. Recombinant hagfish slime threads will be drawn from 25% to 100% with respect to its original length and their properties will be examined.
In case if the linkage of chromoprotein greatly deteriorates the quality of the thread, or leads to self-assembly failure. Chromoproteins will be directly expressed and purified. By mixing different ratios of chromoprotein, solutions of different colours can be obtained. Hagfish slime thread is then directly immersed into the chromoprotein mixture for colouration. This method will be performed in parallel with dyeing the thread with natural dye, such as indigo. Other methods of non-covalently linking chromoproteins to IF will be explored in phase 2.
References
[1] F. Jing, “Biomimetic engineering of materials based on hagfish slime thread proteins,” 2016.
[2] A. Negishi, C. L. Armstrong, L. Kreplak, M. C. Rheinstadter, L.-T. Lim, T. E. Gillis, and D. S. Fudge, “The Production of Fibers and Films from Solubilized Hagfish Slime Thread Proteins,” Biomacromolecules, vol. 13, no. 11, pp. 3475–3482, 2012.