Contents

Preliminary Spider Silk Preparation

Before we began transforming our cells and conducting our synthetic silk experiments, we wanted to begin developing and testing our reduced graphene oxide (rGO) binding protocols since this binding ability is essential to the success of our project. However, since this was occurring before the production of our recombinant silk proteins, we needed to find alternative means for accessing spider silk proteins. With the exorbitant cost of natural farmed spider silk and the ethical issues surrounding it, it was not feasible for our team to purchase natural spider silk, so we turned to collecting wild spider silk around the University of Exeter’s campus.

Since our project aimed to reduce the involvement of animals in the production of our final composite, we decided to avoid collecting webs that were currently occupied by spiders, instead choosing to collect ones that had been previously vacated. However, the silk fibres had become thoroughly contaminated with dust, clothing fibres, dead insects, and other unidentified contaminants, which would likely affect the binding affinity of the fibres, and so our team had to first develop a cleaning protocol that purified the fibres while retaining their structural integrity.

An overview of our cleaning protocols have been detailed below (Table 1).

In our protocol, we have detailed our collection proceedure, the methodology we used when running preliminary cleaning comparisons on the silk samples, and the finalized cleaning protocol we developed based on the results of our preliminary experiments. The detailed protocol can be found by clicking the button below.

CycA characterisation

To identify whether engineering of the alanine permease CycA into E. coli enables elevated net uptake of alanine from growth medium, we determined alanine uptake by BL21 DE3 pLysS E. coli across a 7 hour incubation period, using an alanine detection kit purchased from Merck.

To maximise likelihood of CycA inclusion yielding a detectable increase in alanine uptake, we wanted to determine the maximum concentration of extracellular alanine the growth medium can be doped with without inhibiting E. coli growth significantly. Therefore, we conducted an alanine tolerance preliminary, growing BL21 DE3 pLysS (engineered with either CycA in a pX1900 backbone or with just the plasmid) in alanine concentrations ranging from 5.8 mM (undoped LB) up to 100 mM. We measured OD₆₀₀ and cells/ml every hour across a 7 hour period, determining that extracellular alanine concentrations up to 100 mM do not significantly impact cell growth.

We then determined alanine uptake by BL21 DE3 pLysS, engineered with either CycA in a pX1900 backbone or with just the plasmid, by incubating both strains with and without additional alanine doping of LB (adding 1M alanine solution to make up the growth medium to a final alanine concentration of 100 mM). Once again, we measured OD₆₀₀ and cells/ml every hour across a 7 hour period. Additionally, we harvested pellets from each sample every hour.

Pellets from the t = 3, 5 and 7 hour samples (beginning, middle and end of log phase) were resuspended and their alanine content was determined using the Merck Alanine Assay kit. Alanine uptake is determined via addition of an enzyme which converts present alanine into pyruvate. This pyruvate is bound by a probe, initiating a detectable colour change. Therefore, initial alanine concentration is proportional to absorbance at 570 nm. We then normalised alanine uptake by cells/ml to determine alanine uptake per cell for each sample at the aforementioned timepoints, in order to deduce whether inclusion of CycA elevated rate of alanine absorption. A summary of the alanine detection process is detailed in the figure below. A detailed protocol is available for download by clicking the button below.

Accuracy of silica microsphere OD₆₀₀ calibration

Using OD₆₀₀ as a measurement of population growth for E. coli is inherently limited by the fact that assessing optical density alone cannot distinguish between the effects of live cells and the effects of all other debris on turbidity. Additionally, OD₆₀₀ cannot be used as a measure of live cell count without calibration As investigated in the Interlab Measurement study in 2018, the results of which were outlined in a Beal et al paper^[1], such calibration can be performed using a suspension of silica microsphere beads, allowing OD₆₀₀ to be correlated with beads/ml, in turn giving an approximate measure of cells/ml present in the sample.

Our PI, Dr Chloe Singleton, previously produced a calibration curve across a range of beads/ml concentrations from 0 to 1.5 x 10¹⁰, following the procedure used in the 2018 Interlab study, detailed in the PDF linked below. Using the Imagestream flow cytometry data recorded for normalisation of alanine uptake in CycA characterisation, we were able to directly analyse the accuracy of the estimation of cells/ml from OD measurements using the silica microsphere calibration curve across a 7 hour time course, via comparison with cells/ml data taken directly from sample image analysis. We interpolated the standard microsphere curve, using recorded OD data, to estimate cells/ml and then calculated percentage difference between actual cells/ml data and the estimated values derived from microsphere calibration for all 4 conditions of the alanine uptake analysis. The detailed protocol followed by Dr Singleton, taken from the 2018 Interlab Measurement study can be downloaded by pressing the button below.

Fibroin-rGO Preliminaries

Constant iterations of the design, build, test, learn cycle moulded the following protocols. We have included a PDF to display the progression of our protocols as well as a separate PDF of the final method. After numerous attempts at using silkworm cocoons to make films, we concluded a pre-dispersed medium such as silk fibroin solution would be more suitable for our needs. Methods using both fibroin sources are detailed.

Cocoons:

In regards to composite formation, we carried out preliminary testing of rGO binding with silk fibroin to enable constant progress throughout our project. Fibroin is produced from silkworms rather than spiders, but it is the most available and affordable silk fibre and has previously been investigated for similar applications ^[2] which is why we used it. Our aim was to gain a better insight into the ease of working with rGO and begin to optimise a method for film production.

Initially, silkworm cocoons were used to make a first film as per Liang‘s protocol ^[2]. We purchased silkworm cocoons online which were washed by heating with sodium carbonate in a heat block then undergoing vacuum filtration before washing with MilliQ water. The rGO suspension was added to the sample and ultrasonicated before undergoing vacuum filtration and drying to yield a film.

Conductivity of resulting films (and a baseline for non-rGO combined materials) was measured by the four-pronged probe method that we were informed of by Graphenea. If the sample was not fit for four probe testing, then a multimeter was used. This gave a rough idea of whether conductivity was being affected by treatment of the cocoons.

Solution:

Following early production of films using cocoons, we found it challenging to create an evenly separated solution. We ordered a silk fibroin solution in the hopes of producing a more uniform dispersion of protein and rGO. We used a solution of 5% w/v concentration as this was easily available and similar to concentrations used by previously published literature ^[3].

Butanediol and PVA were later added in this protocol as we found difficulty in producing thermostable composites at room temperature without them. Both additions have good biocompatibility ^[4][5]. Butanediol can induce fibroin to form a crystalline structure which can reduce the dissolution rate of the film and increase its flexibility ^[6]. PVA has an amphiphilic structure which theoretically could aid in dispersing the graphene aqueous solution ^[7]. PVA also increases the young's modulus and yields stress of graphene bionano composites ^[8].

References

• [1] - Beal, J., Farny, N.G., Haddock-Angelli, T. et al. Robust estimation of bacterial cell count from optical density. Commun Biol 200;3(512) Doi: 10.1038/s42003-020-01127-5
• [2] - Liang B, Fang L, Hu Y, Yang G, Zhu Q, Ye X. Fabrication and application of flexible graphene silk composite film electrodes decorated with spiky Pt nanospheres. Nanoscale. 2014;6(8):4264-74.
• [3] - Yusoff NI, Wahit MU, Jaafar J, Wong TW. Characterization of graphene-silk fibroin composites film. Materials Today: Proceedings. 2018 Jan 1;5(10):21853-60.
• [4] Chocarro-Wrona C, de Vicente J, Antich C, Jiménez G, Martínez-Moreno D, Carrillo E, Montañez E, et al. Validation of the 1,4-butanediol thermoplastic polyurethane as a novel material for 3D bioprinting applications. Bioeng Transl Med. 2020 Oct 20; 6(1). Available at https://doi.org/10.1002/btm2.10192
• [5] Khalaji S, Golshan Ebrahimi N, Hosseinkhani H. Enhancement of biocompatibility of PVA/HTCC blend polymer with collagen for skin care application. International Journal of Polymeric Materials and Polymeric Biomaterials. 2021 May 03;70(7):459-468. Available at https://doi.org/10.1080/00914037.2020.1725761
• [6] Zhang H, Zhao J, Xing T, Lu S, Chen G. Fabrication of silk fibroin/graphene film with high electrical conductivity and humidity sensitivity. Polymers. 2019 Oct 28;11(11):1774.
• [7] Ziv E, Attia D, Vasilyev G, Mendelson O, Zussman E, Yerushalmi-Rozen R. The role of polymer–solvent interactions in polyvinyl-alcohol dispersions of multi-wall carbon nanotubes: from coagulant to dispersant. Soft Matter. 2019;15(1):47-54.
• [8] Hong X, Zou L, Zhao J, Li C, Cong L. Dry-wet spinning of PVA fiber with high strength and high Young’s modulus. InIOP Conference Series: Materials Science and Engineering 2018 Nov 1 (Vol. 439, No. 4, p. 042011). IOP Publishing.