Difference between revisions of "Part:BBa K4247029"
(→Protein expression) |
|||
| Line 22: | Line 22: | ||
| − | '''Aim - '''To show | + | '''Aim - '''To show the expression of the MAKI marine-minispidroin protein with a His-tag in the N-terminus. |
| − | '''Results - ''' | + | '''Results - '''Cells expressing MAKI marine-minispidroin proteins with a His-tag in the N-terminus or C-terminus were grown to an OD600 of 0.1 and induced with 0.3mM IPTG to induce protein expression. Then, the cells were grown at 25°C post-induction ON. Further, different amounts of the cell lysate were loaded on the SDS-gel to differentiate the amount of proteins. |
| − | |||
| + | [[File:nhismarine.png]] | ||
| − | '''Conclusion - ''' | + | |
| + | In the lanes of the protein with the His-tag in the C-terminus, we can clearly see a band only in the induced lane at the expected molecular weight. However, this band is absent in the lanes of the protein with the His-tag in the N-terminus. | ||
| + | |||
| + | |||
| + | '''Conclusion - '''Hence, it is evident that when the His-tag is in the C-terminus, the protein is expressed but when the His-tag was switched to the N-terminus, the protein was no longer expressed. | ||
Revision as of 21:30, 9 October 2022
Contents
MAKI_marine_minispidroin_N-6His
This part codes for the full MAKI marine-minispidroin,a chimeric protein formed by combining the sequences of Desis marina, a marine spider's spider silk proteins and minispidroin, a highly soluble spider silk protein. This part, together with BBa_K4247005, BBa_K4247026 and BBa_K4247002 gives the full sequence of the MAKI marine-minispidroin protein.
This part is one of a collection of compatible Marine-minispidroin parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247002 (Minispidroin_CT), BBa_K4247005 (Minispidroin_NT_N-6His), BBa_K4247026 (MAKI_marine_minispidroin_Rep), BBa_K4247027 (MAKI_marine_minispidroin) and BBa_K247028 (MAKI_marine_minispidroin_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.
It has been shown that spider webs from terrestrial spiders undergo structural changes with humidity wherein high humidity causes supercontraction. Supercontraction is a phenomenon where when spider silk is exposed to water, water infiltrates the fibre and causes it to reduce in length to nearly half of it’s length when dry. Major ampullate silk proteins contain a lot of GPGXX motifs wherein G is glycine, P is proline and X can be any amino acid from a small set of amino acids. These motifs form the non-crystalline fractions of the spider silk and when the silk is in a dry state, hydrogen bonds keep these non-crystalline fractions parallel to the fiber axis whereas when the silk is wet, these hydrogen bonds are disrupted which causes a loss of orientation and drives the shrinking and thickening of the fiber.
Hence, supercontraction hinders the use of spiders for underwater applications. However, there are certain spiders in nature that can produce silk in water such as Argyroneta aquatica (freshwater) and Desis marina (marine). Desis marina spiders construct retreats with their silk for protection from tides and pressure. Further, they can trap air in their retreat and remain submerged for up to 19 days. 
Since D.marina’s silk is produced under water, it would be expected that these silks would not supercontract since that is not beneficial for the spiders. Just as expected, a transcriptomics study on D.marina revealed that the silk sequences of D.marina lack the amino acid motifs associated with supercontraction.
It is well known that solubility and pH sensitivity affect the N- and C-terminus which in turn plays a huge role in spinning. So, minispidroin was designed in such a way that it combined the N-terminus and C-terminus from 2 different spiders (E.australis and A.ventricosus) to have high solubility and pH sensitivity to ensure optimal spinning. Considering that the N- and C-terminus have been optimised for spinning, we decided to design a chimeric protein by combining the sequence of D.marina’s MaSp and the minispidroin’s repetitive region (E.australis Masp). This chimeric protein would not only have good solubility and pH sensitivity for optimal spinning but also the ability to persist underwater without undergoing supercontraction.
Herein, part BBa_K247029 codes for the full chimeric protein, MAKI_marine_minispidroin with a 6x His-tag in the N-terminus.
Characterization
Protein expression
Aim - To show the expression of the MAKI marine-minispidroin protein with a His-tag in the N-terminus.
Results - Cells expressing MAKI marine-minispidroin proteins with a His-tag in the N-terminus or C-terminus were grown to an OD600 of 0.1 and induced with 0.3mM IPTG to induce protein expression. Then, the cells were grown at 25°C post-induction ON. Further, different amounts of the cell lysate were loaded on the SDS-gel to differentiate the amount of proteins.
In the lanes of the protein with the His-tag in the C-terminus, we can clearly see a band only in the induced lane at the expected molecular weight. However, this band is absent in the lanes of the protein with the His-tag in the N-terminus.
Conclusion - Hence, it is evident that when the His-tag is in the C-terminus, the protein is expressed but when the His-tag was switched to the N-terminus, the protein was no longer expressed.