Patent Application: US-201013499890-A

Abstract:
described are silk proteins derived from spider mite , more specifically derived from tetranychus urticae . more specifically , described is the use of these proteins to make fibers , or fiber - composed material and the resulting fibers and materials .

Description:
the london population of t . urticae developed from the isofemale line in london ontario , following eight backcrosses ( to generate maximum homozygote population ) was mass produced on the bean plants in growth chambers at 27 ° c . and 16 : 8 photoperiod . plants were washed in 0 . 1 % triton x detergent solution in 2 - liter beakers to release all spider mite life stages . adult spider mites , nymphs , larvae and eggs were filtered through series of fine sieves to isolate pure egg fraction . eggs were collected in the eppendorf tube , treated with bleach solution ( to remove plant tissue and possible microbial contaminants ) and prepared for the dna extraction . embryos were ground in the glass tissue grinder and dna extraction was performed using quiagen blood & amp ; cell culture dna kit ( midi column # 13433 ) according to manufacturer &# 39 ; s protocol . dna for whole genome sequencing project was sequenced using sanger sequencing protocol at the joint genome institute ( usa department of energy ), walnut creek , calif . from fragments of fibroin genes available in the database , blastp and tblastn were run over the proteome and genome of tetranychus urticae . the obtained hits were all checked manually as due to the highly repeated nature of the sequence problems occurred with the prediction and even assembly of the original genomic sequence . about half of the gene models were originally wrongly predicted , involving incorrectly predicted reading frames . the corrections were iteratively evaluated and aligned using muscle , including the existing fibroin genes from the public databases and the already found ( and corrected ) genes found in tetranychus urticae . the originally found proteins all had in common a high percentage of g , a and p organized in repetitive patterns . this particular aspect was further used to identify more divergent proteins having similar patterns . to find them , tblastn was run again with the low - complexity filters turned off . from the multiple hits returned , six more genes were retained , based on similarity of patterns and coverage by illumina transcript reads . all were manually annotated and added to the already found genes , as potentially involved in the fibers . in total , twelve genes were found having a similar repetitive domain . mechanical and antimicrobial characteristics of the spider mite silk are investigated . thread thickness and strength are measured using the standard techniques . the favimat - robot ( textechno ) is used to analyze the tensile properties . it is a semi - automatic single - strength tester , working according to the principle of constant rate of extension ( din 51221 , din 53816 , iso 5079 ). the instrument is equipped with a balance allowing the mass to be measured at a high resolution of 0 . 1 mg . the instrument includes a robot , which is a fiber storage , equipped with a computer - controlled transfer clamp for the transport of the single fiber to the testing position of the favimat . moreover , this instrument is equipped with an integrated measuring unit for linear density ( in dtex = 0 . 1 g / km ). this has the considerable advantage , certainly for natural fibers , that the fineness is determined simultaneously with the tensile properties . the linear density is measured according to the vibroscopic method ( astm d 1577 — bisfa 1985 / 1989 chapter f ). the fiber is preloaded at a predefined speed . further on , the fiber is subjected to an electro - acoustic sinusoidal vibration and the resonance frequency is detected with an opto - electronic sensor . the fiber linear density is calculated from the resonance condition , i . e ., length , preload , and resonance frequency of the fiber . suggesting a uniform mass distribution and a round cross - section , the linear density can be calculated as follows : in this equation , tt is the linear density in dtex , fv is the preload in cn , f is the resonance frequency and l is the test length in mm . as spider mite silk is very resistant to degradation , possible antimicrobial activity of the silk is measured by measuring the inhibition circle around the silk on solid medium confirmation of the presence of the proteins in spider mite silk by mass spectrometry ( ms ) analysis ten t . urticae adults were placed into capped and parafilm - sealed 35 mm petri plates for 24 hours at room temperature . petri plate cap was removed and examined for signs of mites , eggs and debris , which were removed as necessary . after this , a cap was washed with 1 ml of 95 % ethanol and silk threads suspended in ethanol were collected in eppendorf tubes . content of 10 - 15 tubes was pooled together and silk threads were transferred to a glass container for a wash with acetic acid . silk threads were transferred back into 95 % ethanol , pulled apart , and transferred into eppendorf tubes with 95 % ethanol for storage and subsequent analysis . silk thread suspensions were initially evaporated using a speedvac system . the dried samples were re - suspended in 75 % tfa ( trifluoroacetic acid ) in glass vials . vials were then microwaved for 45 minutes at full power in a beaker filled with water . the contents of the vials were then dried using a speedvac system and , following this , reconstituted in 10 % formic acid . samples were then injected on a q - tof ms system using a 150 minute 0 - 40 % acn gradient acquiring data in a data - dependent fashion . data analysis was performed using peaks studio 5 . 2 software . peptides were matched against t . urticae proteome database . analysis was performed both with and without consideration for possible variable post - translational modifications , such as deamidation and oxidation . protein id matches from t . urticae proteome database that appeared in both types of analysis and were also predicted using computational approach were considered for subsequent amplification and cloning by means of pcr . seq id no : 3 , seq id no : 14 , and seq id no : 17 have been confirmed as being part of the silk by ms . use of the polymerase chain reaction ( pcr ) to confirm gene expression t . urticae rna was extracted using trizol reagent ( invitrogen ). samples for pcr were prepared by reverse transcribing 3 μg of total rna using superscript ii reverse transcriptase ( invitrogen ). aliquots of this reaction were then used in pcr reactions . primers for pcr were designed to amplify short ( 100 - 200 bp ) fragments from the non - repetitive 5 ′ and 3 ′ regions of candidate genes predicted mrna sequence . pcr was performed using taq dna polymerase ( fermentas ) according to manufacturer &# 39 ; s recommendations and amplified fragments were cloned into pgem - 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