Part:BBa_K2647004
PrancerPurple linked to 6 silk repeats and a cellulose binding module
This part comprises of chromoprotein PrancerPurple linked to 6 repetitive sequences of spider dragline silk ADF-3 from European garden spider Araneus diadematus and cellulose binding module from Clostridium thermocellum. Silk repeats are flanked with 23 amino acid long naturally occurring N- and C-terminal linkers, derived from major ampullate spidroin 1 from Euprosthenops australis. Linkers act to prevent the disordered structure of the silk repeats to disturb the folding of the chromoprotein and the binding domain.
Contents:
1. Usage and Biology
2 Large Scale Production
2.1 Cultivations and Induction
2.2 Cell Lysis and Purification
3. Concentration process
4. Cellulose binding
1. Usage and Biology
Spider dragline silk is extremely strong and elastic making it tougher than most of the natural or manmade fibers. This construct code protein which could be used for forming pre-dyed silk fibers or to be used as a coating for keratin based materials.
PrancerPurple and silk are stable in higher temperatures as well as KBD based on our modeling results which enables this construct to be lysed using heat which can lower the production costs.
PrancerPurple, CBM and silk are stable in higher temperatures which enables this construct to be lysed using heat which can lower the production costs.
2 Large Scale Production
2.1 Cultivation and Induction
In order to get enough material for our tests we started production in large scale. Protein expression was started by inoculating 1-2 colonies of expression strain cells which were transformed with plasmids containing our gene of interest to 5ml of LB medium with 50ug/ml Kanamycin and incubated at + 37°C with shaking over night. This starting cultivation was then inoculated to 45ml of LB medium with 50ug/ml Kanamycin and incubated at +37°C with shaking for ~7 hours until the OD600 value was between 0.6-0.8. The whole 50ml was then inoculated to 450ml of LB medium with 50ug/ml Kanamycin in 1l and induced at the same time with final concentration of 0.5mM of IPTG. The flask was sealed with air permeable membrane and inoculated at +30°C with gentle shaking over the weekend.
2.2 Cell Lysis and Purification
Cells were harvested next day by transferring the culture to sorvall centrifuge bottle and centrifuged at 10 000 for 30 minutes. Supernatant was discarded and the pellet was resuspended in 10ml of lysing mix (DNAse1, Lysozyme, 5mM magnesium chloride, 50mM Trish-HCL pH 7.4, protease inhibitor) for 1g of cell pellet and incubated at room temperature for 30 minutes. Cells were sheared using sonification after which the sample was incubated at +70°C waterbath for 30 minutes. Because the silk is unstructured it does not denaturate and both PrancerPurple and CBM are heat resistant we can denature all the other soluble proteins with heat and we are left with our protein in the supernatant when centrifuged at 10 000 for 90 minutes.
3. Concentration process
The protein was concentrated with Econo-Pac® 10DG Desalting Prepacked Gravity Flow Columns. In principle, when the silk protein is concentrated silk repeats interact with each other forming a fiber. We tried to make fibers by inserting tweezers in our concentrated solution and carefully opening them. We added a drying step by using hot air, to further concentrate the solution.
4. Cellulose binding
The cellulose binding module was tested by binding the proteins to nano cellulose and checking the result on SDS-page . As cellulose we used nanofibrils (CNF) derived from birch. 1%(w/v) nanocellulose solutions were prepared from stock concentration and binding test was conducted to different concentrations of proteins (1x, 10x and 100x). CNF and protein mixtures were incubated for half an hour at room temperature after which they were centrifuged for 5 min at 4000g. CNF and protein bound to it were pelleted at the bottom and all the non-bound protein was found in the supernatant. Samples from supernatants were loaded to SDS-PAGE gels and from the gel it could be seen that bands are missing from the lanes 7 and 8 which implies that protein has bound to CNF.
References
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 693
Illegal BsaI.rc site found at 1512
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