As we have known for a long time, nature is an excellent source of inspiration for many studies, discoveries and experiments. Birds and winged insects have shown us that the sky is quite achievable, aquatic mammals have shown us how to prolong our stay under water, and spiders have proved that even the smallest creatures can create something incredible. In the study we are considering today, scientists from the Institute for Physical and Chemical Research (Wako, Japan) have found a way to create an artificial web using phototrophic bacteria. How exactly did they achieve this, how natural the resulting spider webs are and why were phototrophic bacteria used? Answers to these and other questions await us in the report of scientists. Go.
Research basis
For many people, spiders are "oh God, get him off me!" or "ugh, what disgusting." However, setting aside phobias and prejudice against these creatures, one can consider how unique these eight-legged predators are. Spider anatomy is literally made for perfect hunting. On the one hand, there is a poison that can paralyze or even kill prey or the enemy. On the other hand, there are well-developed sense organs (especially touch due to trichobothria (hairs) throughout the body). The visiting card of spiders, which has become the basis for many metaphors and catchphrases, is their web.
A video about how spiders produce their famous web.
At its core, a web is a protein, the composition of which is rich in glycine (C 2 H 5 NO 2 ), alanine (NH 2 -CH (CH 3 ) -COOH) and serine (HO 2 C-CH (NH 2 ) CH 2 OH ). A cobweb is formed in a special gland, where it is in liquid form.
When the secretion of the gland is secreted out through numerous spinning tubes, special spider warts form threads from it, which the spider uses to build its deadly traps.
Spider web threads are unique because their mechanical properties are superior to many other materials. For example, the tensile strength of a spider web for an ordinary spider ( Araneus diadematus ) is 1.1-2.7 GPa, for a human hair it is 0.25 GPa, and for steel it is 0.4-1.5 GPa. The density of spider silk is 1/6 that of steel (1.3 g / cm 3 ). That is, if you go around the Earth with a web, then its weight will be only 500 grams. The energy density of about 1.2 × 108 J / m 3... Also, spider silk is extremely flexible, i.e. can stretch up to 5 times its original length (in a relaxed state) without any breaks. The impact strength of the spider web is comparable to polyaramide yarns. Spiders are not extremophile, but their webs can easily survive temperatures from -40 ° C to 220 ° C. In addition, the biodegradable and biocompatible properties of silk make it suitable for medical applications.
Comparison of tensile strength of steel thread and spider silk (corresponding density).
It is also curious that one type of spider can produce several types of webs that are different in their properties and applications (spiders of the Argiope argentata species produce as many as 5 variants of the web):
- for the outer edges and web frame (very durable);
- for areas of prey capture (very sticky, elastic and durable);
- ( );
- ( 2 3 );
- ;
- .
This is only a brief description of spider silk, but even that is enough to understand the uniqueness of this substance. That is why many scientists have been trying for a long time to create an artificial equivalent of the web. For many, this is quite successful. However, the problem of mass production remains unsolved.
In the work we are considering today, scientists have proposed a solution to this problem. It consists in using the purple bacterium Rhodovulum sulfidophilum , which has the properties of both phototrophs * and halophiles * .
Phototrophs * are organisms that use light to generate energy.
Halophiles * are a type of extremophile that live in high salinity conditions.R.sulfidophilum is a marine anoxygenic photosynthesizing * bacterium with different metabolic characteristics that produces biohydrogen, bioplastics and extracellular nucleic acids.
Anoxygenic photosynthesis * - unlike conventional photosynthesis, molecular oxygen is not formed during anoxygenic photosynthesis.But the most important skill of R.sulfidophilum for scientists is its ability to grow in photoautotrophic conditions through the use of inexpensive and renewable resources such as light (energy), CO 2 (carbon source) and N 2 (nitrogen source) through the processes of photosynthesis and fixation. nitrogen. In addition, R.sulfidophilum thrives in seawater, which helps to reduce the risk of biological contamination during cultivation.
In recent years, good results have been achieved in mass production of spidroin (MaSp, spider silk protein) using recombinant host organisms ( Escherichia coli bacteria , Pichia pastoris yeast , silkwormBombyx mori , tobacco, mammalian cell cultures, etc.).
The authors of this work do not deny the success of their predecessors, but note the extremely small volumes of production and the rather high cost of the final product due to the high cost of the production itself (in the case of microbial fermentation, 70% of the production costs are raw materials).
In their study, the authors propose a new method for the economical and efficient production of spider silk using the bacterium R.sulfidophilum , capable of producing a hydrophobic repeat sequence MaSp1 (spidroin-1) using a small amount of organic matter under conditions of photoheterotrophic or photoautotrophic growth.
Research results
First it was necessary to prepare the bacteria R.sulfidophilum .
It has previously been reported about the possibility of introducing exogenous plasmid DNA into R.sulfidophilum by bacterial conjugation * using plasmids derived from pCF1010 and E. coli S17-1 as a donor strain.
Conjugation * - unidirectional transfer of a part of the genetic material during direct contact of two bacterial cells.In this study, it was decided to use a different vector (pBBR1MCS-2) containing a kanamycin resistance gene, a mob gene encoding a specific nuclease, and a transfer source ( oriT * ), which were widely used in gram-negative bacterial conjugation.
oriT * is a short sequence (up to 500 bp) necessary for the transfer of DNA containing it from a bacterial host to a recipient during bacterial conjugation.In the chromosome of R.sulfidophilum (accession number NZ_CP015418), two tellurite resistance genes encoding a tellurite resistance protein of the TerB family were present in the A6W98_RS06280 and A6W98_RS17070 loci.
Traits of resistance to kanamycin and tellurite were used as markers for the selection of positive conjugants of R. sulfidophilum .
Plasmid * pBBR1-Ptrc-MaSp1 contained:
- the * trc promoter (Ptrc), which is a potent hybrid constitutive promoter in E. coli;
- the sequence "AGGAGA" of the ribosome binding region (RBS);
- MaSp1 Nephila clavipes, ( ) E.coli (1a, 1b).
* — , .
* — , - ( ).Approximately 0.4 g of wet cell mass (CWM from cell wet mass ) was obtained from 50 ml of recombinant R. sulfidophilum culture grown to stationary growth under photoheterotrophic conditions, namely from sea broth (MB from marine broth ) with LED illumination at (730 nm, 20-30 W / m 2 ) for 4 days.
Image # 1
Despite the fact that overexpression of the recombinant MaSp1 proteins was not clearly detected in all recombinant cultures of R. sulfidophilum using polyacrylamide gel electrophoresis / SDSPAGE ( 1c), the positive expression of MaSp1 proteins was confirmed by the protein immunoblot method for all created recombinant R.sulfidophilum cells carrying pBBR1-Ptrc-MaSp1- (1-mer, 2-mer, 3-mer, or 6-mer) ( 1d ).
K-measures * - subsequences of length k contained in a biological sequence.The only repeat domain in the resulting constructs contains 33 amino acid residues of the following form:
NH 2 -SGRGGLGGQGAGAAAAAGGAGQGGYGGLGSQGT-COOH.
The theoretical molecular weight of target proteins, including nonsidroin sequences (cleavage regions of His-Tag, S-Tag, enterokinase and thrombin) at the N-terminus is 7.9 kDa for 1-mer (81 aa); 10.5 kDa for 2-mer (114 aa); 13.1 kDa for 3-mer (147 aa) and 20.9 kDa for 6-mer (246 aa).
Yes - designation of the Atomic mass unit; kDa - kilodalton (1 kDa = 103 Da).In addition to confirming the expression of MaSp1 proteins, their amount of MaSp1 proteins obtained from recombinant cultures of R.sulfidophilum was also assessed : ~ 3-10 mg / L (1-mer = 3.4 mg / L; 2-mer = 3.9 mg / L ; 3-mer = 10.2 mg / l; 6-mer = 6.8 mg / l) or 3.5–6.9% of the total amount of proteins. For comparison, heterologous expression of spidroins in the well-known and widely used recombinant system of E. coli is capable of producing only ~ 0.3–1.2 mg / L of purified spidroin.
The dominant allele is indicated by a capital letter (A versus a). Since each parent provides one allele, the following combinations are possible: AA, Aa, and aa.
The most surprising result, according to scientists, in this study is the demonstration of microbial cell factories, based on marine photosynthetic organisms, in which a photoautotrophic growth regime can be applied using renewable non-food raw materials and seawater as a culture medium.
R.sulfidophilum , carrying pBBR1-Ptrc-MaSp1- (6-mer), was cultured in artificial sea water (Daigo ASW from artificial seawater) when illuminated by LEDs (730 nm, 20-30 W / m 2 ) with the bicarbonate salt (1 g / l) as a source of inorganic carbon and nitrogen gas (0.5 l / d) as a source of nitrogen. The cultivation time was 7 days ( 2a ).
Image No. 2
The largest constituent unit, MaSp1- (6-mer), was chosen for subsequent experiments because the higher molecular weight of MaSp1 would result in a higher tensile strength of the spider silk fiber. Sodium bicarbonate has been used to supply inorganic carbon because bicarbonate salts have higher solubility and lower transportation costs than CO 2 gas .
Previous researchers have carried out experiments to determine the required lighting condition for the growth of cells R.sulfidophilum : intensity (8 and 50 W / m 2 ) and the wavelength (730, 800 and 850 nm). This study evaluated the growth effect of recombinant R.sulfidophilumseveral additional nutrients (yeast extract, vitamin, iron and phosphorus) that are deficient in ASW media.
Dry cell mass (CDM) decreased from 0.90 g / L (with all nutrients) to 0.66 g / L in the absence of yeast extract and to 0.39 g / L in the absence of phosphorus. It has also been determined that recombinant R.sulfidophilum cannot grow in ASW without NaHCO 3 , N 2 gas, or phosphorus ( 2b ; ASW + N 2 , ASW + C + N 2 , ASW + C + P, and ASW + P + N 2 ).
CDM (~ 0.4 g / L) in such ASW medium variants most likely originated from inoculants *or inoculum even after samples have been washed with 2% sodium chloride. Thus, sources of carbon, nitrogen, and phosphorus are required for the growth of recombinant R.sulfidophilum in ASW medium.
Microbiological inoculant * - biological products containing live cultures of microorganisms useful for plants.As expected, cell growth significantly increased from 0.34 ± 0.02 g / L (ASW + C + N 2 ) to 0.58 ± 0.08 g / L (1.7 times increase) and 0.81 ± 0.02 g / L (2.4 times increase) in the presence of yeast extract (ASW + C + N 2 + YE) and phosphorus (ASW + C + N 2 + P), respectively.
The highest CDM was achieved by adding yeast extract and phosphorus, giving 1.04 ± 0.06 g / L (a 3.1-fold increase).
The yield of ~ 0.2 mg / L of recombinant protein MaSp1 (2% of the total amount of proteins) was observed under the conditions of ASW + N 2 , ASW + C + N 2 , ASW + C + P and ASW + P + N 2 ( 2c and 2d). The production of the MaSp1 protein was aided by the addition of a yeast extract, which significantly increased the yield of the MaSp1 protein from 0.12 ± 0.10 mg / L (ASW + C + N 2 ) to 3.93 ± 2.76 mg / L (ASW + C + YE + N 2 ).
The addition of yeast also increased the percentage of MaSp1 in total proteins from 1.2 ± 1.0 to 6.9 ± 5.3%. Interestingly, the addition of phosphorus, while positively affecting the increase in CDM, negatively affected the production of the MaSp1 protein.
Compared to ASW + C + YE + N 2 in the ASW + C + YE + P + N 2 medium, the yield of the MaSp1 protein decreased to 2.71 ± 1.09 mg / L, and the percentage of MaSp1 in total proteins decreased to 3.9 ± 1.6%.
The explanation for these differences in protein production depending on the culture medium may lie in the functionality of each of the components. For example, yeast extract is primarily a nitrogen source that promotes protein biosynthesis. Meanwhile, phosphorus is an important macronutrient and hetero-element in many important cellular compounds, which promotes the growth of primary producers.
To get spider silk in its usual form, it was necessary to purify MaSp1.
Image # 3
To obtain enough MaSp1 protein for fiber extrusion, fermentation ( 3a ) was carried out to produce MaSp1- (6-mer).
In general, the size of spidroins is positively correlated with tensile strength to a specified molecular weight. Larger proteins have more interchain and intrachain interactions, more entanglements, and fewer end-chain defects.
Purification of MaSp1- (6-mer) was performed using affinity chromatography through a histidine tag, which was present at the N-terminus of the MaSp1 gene cassette, and gel filtration chromatography. As a result, ~ 10 mg of purified MaSp1- (6-mer) ( 3b ) was obtained from ~ 40 g of CWM.
Silk fibers were prepared by pipetting 10 wt% purified MaSp1- (6 measure) dissolved in hexafluoroisopropanol (HFIP) into a coagulation bath followed by manual pulling with forceps ( 3c). The best results were obtained using 90% 2-propanol as the coagulation bath, which caused relatively gentle dewatering, which allowed for efficient yarn pulling. Analysis by scanning electron microscopy showed that the fibers have a diameter of 10–20 µm and a surface covered with grooves parallel to the fiber axis. Fractography showed that the internal structure consisted of microfibrils ( 3d and 3e ).
For a more detailed acquaintance with the nuances of the study, I recommend that you look into the report of scientists and additional materials to it.
Epilogue
In this work, scientists talked about the successful creation of a micro-factory for the production of the MaSp1 protein. The main leader of this production is the bacterium R.sulfidophilum , thanks to which it was possible to achieve photoheterotrophic expression of an artificial spider web protein under conditions of photoautotrophic growth.
In other words, scientists have genetically modified the bacterium to produce spider silk, more specifically the protein MaSp1, which is an important component of it. In addition to genetic manipulations, it was also necessary to establish the optimal conditions for the cultivation of bacteria. As it turned out, the ideal environment is artificial seawater. Additionally, nitrogen gas and yeast extract had to be used as nutrient sources. Together, these constituents lead to efficient bacterial growth and therefore the production of spider web protein.
It is also important that the spider web fibers obtained from the protein are very similar in structure to the natural ones produced by spiders of the Nephila species .
The scientists note that their method of growing spider silk protein can be used to grow other substances as well. In the future, the authors of the study plan to improve their micro-factory to increase the volume of protein production and improve the molecular characteristics of the resulting product.
According to scientists, their work can contribute to solving many problems: energy, water and food crises, problems with solid waste, global warming, etc. The reason for this wide range of possibilities is the fact that factories like these produce biodegradable and biocompatible materials using a carbon neutral process.
Thanks for your attention, stay curious and have a good work week, guys. :)
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