Difference between revisions of "Part:BBa K4247011"
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'''Results - '''Cell cultures were grown ON at 37°C. Then, the next day, the cultures were diluted to an OD600 of 0.1 and induced with 0.1, 0.3, 0.5 and 1mM IPTG and grew ON. We can clearly see that around 40kDa, there is a darker band in the induced lanes compared to the uninduced lane, showing that the protein is expressed upon induction with IPTG. Further, among the induced lanes, protein expression seems to be best in the range of 0.1-0.5 mM, but a western blot is needed for any clear conclusion. | '''Results - '''Cell cultures were grown ON at 37°C. Then, the next day, the cultures were diluted to an OD600 of 0.1 and induced with 0.1, 0.3, 0.5 and 1mM IPTG and grew ON. We can clearly see that around 40kDa, there is a darker band in the induced lanes compared to the uninduced lane, showing that the protein is expressed upon induction with IPTG. Further, among the induced lanes, protein expression seems to be best in the range of 0.1-0.5 mM, but a western blot is needed for any clear conclusion. | ||
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Revision as of 15:40, 28 September 2022
Contents
Minispidroin_NT-4rep-CT
This composite part codes for the full minispidroin protein, a highly soluble spider silk protein. This is a composite part consisting of the following basic parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_2rep) and BBa_K4247002 (Minispidroin_CT). BBa_K4247004 contains the coding sequence for the full minispidroin protein with 2 repeats of the central repetitive domain.
This part is one of a collection of compatible minispidroin parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_2rep), BBa_K4247002 (Minispidroin_CT), BBa_K4247004 (Minispidroin_NT-2rep-CT), BBa_K247005 (Minispidroin_NT_N-6His), BBa_K247007 (Minispidroin_NT-2rep-CT_N-6His), BBa_K247010 (Minispidroin_NT-2rep-CT-SnoopTag_N-6His), BBa_K247011 (Minispidroin_NT-4rep-CT), BBa_K247012 (Minispidroin_NT-4rep-CT_N-6His), BBa_K247013 (Minispidroin_NT-4rep-CT-SnoopTag_N-6His).
Usage and Biology
Dragline silk produced by spiders is one of the strongest natural materials to exist and it is mainly made up of structural proteins called spidroins. These spidroins consist of non-repetitive N-terminal and C-terminal domains and a repetitive central part consisting of tandem repeats of a certain amino acid sequence. These sequences are rich in alanine and glycine to form the crystalline and amorphous parts of the fibre respectively.
There are many research articles whose authors could successfully produce recombinant spider silk proteins and spin them into fibres by mimicking the conditions of the spider’s silk gland where the fibers are formed naturally. But a major drawback in many of these recombinant spidroins was their low solubility. It has been found that the N-terminus of the spidroin is highly soluble at neutral pH which contributes to the solubility of the protein.
In the spider's silk gland, before spinning, the spidroins remain in a highly concentrated and soluble state. Then, this highly concentrated spidroin solution called spinning dope is subject to a gradual drop in pH from 7.6 to 5.7 along the gland which triggers the formation of the fiber. This drop in pH triggers the N-terminus to be more stable and form large network-like structures whereas the C-terminus becomes more unstable to drive spontaneous fibre formation by forming the beta-sheet fibrils which form the core of the fiber. The N-terminal domain restricts the formation of silk fibers to a precise point in the silk duct, preventing silk proteins stored in the silk gland from agglutinating.
This clearly shows us that the solubility and pH sensitivity have a huge effect on the N- and C-terminus of the spidroin which thus affects the formation of fibers. It has been found that the N-terminus of MaSp1 (Major ampullate spidroin 1) from Euprosthenops australis, shows extremely high solubility and pH sensitivity whereas the C-terminus has low solubility and is inert to pH changes and vice versa for the MiSp (Minor ampullate spidroin) of Araneus ventricosus.
Herein, part BBa_K4247011 is a composite part formed from the following basic parts: BBa_K4247000 (Minispidroin_NT), 2x BBa_K4247001 (Minispidroin_2rep) and BBa_K4247002 (Minispidroin_NT). BBa_K4247003 contains the coding sequence for the full minispidroin protein with 4 repeats of the central repetitive domain.
Characterization
Optimization of inducer concentration
Aim - To determine the concentration of inducer required for optimal protein expression, the weight of the desired protein is 40.3 KDa.
Results - Cell cultures were grown ON at 37°C. Then, the next day, the cultures were diluted to an OD600 of 0.1 and induced with 0.1, 0.3, 0.5 and 1mM IPTG and grew ON. We can clearly see that around 40kDa, there is a darker band in the induced lanes compared to the uninduced lane, showing that the protein is expressed upon induction with IPTG. Further, among the induced lanes, protein expression seems to be best in the range of 0.1-0.5 mM, but a western blot is needed for any clear conclusion.
