Difference between revisions of "Part:BBa K3264013"
| Line 26: | Line 26: | ||
===Purification and SDS PAGE=== | ===Purification and SDS PAGE=== | ||
| − | [[File: 2repctsfgfpfig1.png|center]] | + | [[File: 2repctsfgfpfig1.png|center|500px]] |
| − | [[File: 2repctsfgfpfig2.png|center]] | + | [[File: 2repctsfgfpfig2.png|center|500px]] |
In order to detect whether protein expression was induced by adding isopropylthiogalactoside (final concentration 0.3mM), we used SDS-page(10%) to determine the presence of target protein. Target protein after purification by Ni-NTA method was compared to their original chromoprotein only. Results showing the size chromoprotein only, 2Rep-chromoprotein, and NT-2Rep-chromoprotein are in an increasing trend as regions are added, this result is constant in all three of our chromoprotein. This suggests that our 2Rep protein domain or NT-2Rep region is successfully added on to our pure chromoprotein and well induced. | In order to detect whether protein expression was induced by adding isopropylthiogalactoside (final concentration 0.3mM), we used SDS-page(10%) to determine the presence of target protein. Target protein after purification by Ni-NTA method was compared to their original chromoprotein only. Results showing the size chromoprotein only, 2Rep-chromoprotein, and NT-2Rep-chromoprotein are in an increasing trend as regions are added, this result is constant in all three of our chromoprotein. This suggests that our 2Rep protein domain or NT-2Rep region is successfully added on to our pure chromoprotein and well induced. | ||
Placing the three kinds of chromoprotein, each of them with two kinds of improvements to compare. All 2Rep-chromoprotein under white light source have the most opacity in their own group of chromoprotein of same color. All NT-2Rep-chromoprotein is translucence. However, whereas sfGFP and eforRed chromoprotein only are relatively transparent to all other in their roll, amilCP chromoprotein only self-assembles and gradually subside. The precipitates of amilCP chromoprotein can be dissolved into the solution again after a small shake but reforms in a short period of time. | Placing the three kinds of chromoprotein, each of them with two kinds of improvements to compare. All 2Rep-chromoprotein under white light source have the most opacity in their own group of chromoprotein of same color. All NT-2Rep-chromoprotein is translucence. However, whereas sfGFP and eforRed chromoprotein only are relatively transparent to all other in their roll, amilCP chromoprotein only self-assembles and gradually subside. The precipitates of amilCP chromoprotein can be dissolved into the solution again after a small shake but reforms in a short period of time. | ||
===Fiber spinning of NT2RepCT with recombinant chromoprotein=== | ===Fiber spinning of NT2RepCT with recombinant chromoprotein=== | ||
| − | [[File: 2repctsfgfpfig3.png|center]] | + | [[File: 2repctsfgfpfig3.png|center|500px]] |
Recombinant chromoprotein 2Rep-chromoprotein concocting with NT-2Rep-CT spidroin in the suitable ratio can form the most continuous and stable silk when spinning into 100%isopropanol comparing with the silk spun by replacing 2Rep-chromoprotein with chromoprotein only or NT-2Rep-chromoprotein. It’s also the most transmittance | Recombinant chromoprotein 2Rep-chromoprotein concocting with NT-2Rep-CT spidroin in the suitable ratio can form the most continuous and stable silk when spinning into 100%isopropanol comparing with the silk spun by replacing 2Rep-chromoprotein with chromoprotein only or NT-2Rep-chromoprotein. It’s also the most transmittance | ||
The stable feature of this particular silk spun supports our assumption to let the 2Rep region bounded to the chromoprotein assist the NT-2Rep-CT extensive region’s transition into beta-sheet formation as the 2Rep regions can lie parallel to each other and be formed into a beta-sheet structure cohesively without disruption to the dimer forming at NT or the amyloid-like fibril forming at CT when carrying out artificial silk spinning at low pH or in 100% isopropanol. | The stable feature of this particular silk spun supports our assumption to let the 2Rep region bounded to the chromoprotein assist the NT-2Rep-CT extensive region’s transition into beta-sheet formation as the 2Rep regions can lie parallel to each other and be formed into a beta-sheet structure cohesively without disruption to the dimer forming at NT or the amyloid-like fibril forming at CT when carrying out artificial silk spinning at low pH or in 100% isopropanol. | ||
| Line 39: | Line 39: | ||
===Scanning electron microscopy of fibers=== | ===Scanning electron microscopy of fibers=== | ||
| − | [[File: 2repctsfgfpfig4.png|center]] | + | [[File: 2repctsfgfpfig4.png|center|500px]] |
| − | [[File: 2repctsfgfpfig5.png|center]] | + | [[File: 2repctsfgfpfig5.png|center|500px]] |
Aiming to verify the distribution uniformity of the mixture of recombinant chromoprotein and spidroin after spinning into fiber, we looked at our artificial spun silks under electron microscope. AmilCP is not scanned due to its lack of fluorescence. We randomly selected the cross section in our continuous silk, it has clearly shown that the fiber circularity is qualified as a uniformly distributed silk. | Aiming to verify the distribution uniformity of the mixture of recombinant chromoprotein and spidroin after spinning into fiber, we looked at our artificial spun silks under electron microscope. AmilCP is not scanned due to its lack of fluorescence. We randomly selected the cross section in our continuous silk, it has clearly shown that the fiber circularity is qualified as a uniformly distributed silk. | ||
For both sfGFP and eforRed, when 2Rep is added to the front of chromoprotein, the silk formed is has the smallest diameter among all the silks in the chromoprotein same group. | For both sfGFP and eforRed, when 2Rep is added to the front of chromoprotein, the silk formed is has the smallest diameter among all the silks in the chromoprotein same group. | ||
Revision as of 16:49, 21 October 2019
2Rep-sfGFP
Spider silk serves as a new material with superior properties that can be applied in medication, cloth, and aerospace fields. However, spider breeding is not applicable due to spider's fierce behavior. The current approach is to produce recombinant spidroins (silk proteins) from other chassis and spin them into silk. We here use sfGFP combined with the an extensive repetitive region-Rep
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 SapI.rc site found at 292
Usage and Biology
2Rep-sfGFP is the recombinant chromoprotein that can mix with the NT-2Rep-CT spidroin in a certain ratio to be artificially spun into the continuous and high function spider silk with green fluorescence property. This year, we synthesized the part NT-2Rep-CT, to for artificial silk spinning, is the common architecture in two main types of spider silk proteins: major ambulate spidorins(MaSps) and minor ampullate spidorins (MiSps): A non-repetitive N-terminal domain (NT), as well as the C-terminal domain (CT). In between them is an extensive repetitive region (Rep). We believe this part can realize the approach of producing recombinant spidroins (silk proteins) from other chassis and spin them into silk to fulfill the demand of spider silks. However, this part is only responsible for to form continuous silks that are colorless. In order to reach a larger range of functions, and apply the artificial spun silk to a wider range of industries such as the clothes industry. This year, we also constructed a part collection of recombinant chromoprotein based on three previous parts: sfGFP (BBa_xxxxxx), eforRed (BBa-xxxxxxx), and amilCP(BBa-xxxxxxx). Two kinds of improvements are applied to the original chromoprotein: adding a 2Rep region, or adding NT domain, followed by 2Rep region in the front of the nucleotide sequence of chromoprotein. In this part, we added the 2Rep region in front of chromoprotein sfGFP (BBa_xxxxxxx) to form the recombinant chromoprotein.
Reference
Characterization
We aimed to use a synthetic biology approach to combine a standardized DNA part 2Rep-sfGFP, and transform constructs to metabolically engineered Escherichia coli (E.coli) for bioproduction.
Purification and SDS PAGE
In order to detect whether protein expression was induced by adding isopropylthiogalactoside (final concentration 0.3mM), we used SDS-page(10%) to determine the presence of target protein. Target protein after purification by Ni-NTA method was compared to their original chromoprotein only. Results showing the size chromoprotein only, 2Rep-chromoprotein, and NT-2Rep-chromoprotein are in an increasing trend as regions are added, this result is constant in all three of our chromoprotein. This suggests that our 2Rep protein domain or NT-2Rep region is successfully added on to our pure chromoprotein and well induced. Placing the three kinds of chromoprotein, each of them with two kinds of improvements to compare. All 2Rep-chromoprotein under white light source have the most opacity in their own group of chromoprotein of same color. All NT-2Rep-chromoprotein is translucence. However, whereas sfGFP and eforRed chromoprotein only are relatively transparent to all other in their roll, amilCP chromoprotein only self-assembles and gradually subside. The precipitates of amilCP chromoprotein can be dissolved into the solution again after a small shake but reforms in a short period of time.
Fiber spinning of NT2RepCT with recombinant chromoprotein
Recombinant chromoprotein 2Rep-chromoprotein concocting with NT-2Rep-CT spidroin in the suitable ratio can form the most continuous and stable silk when spinning into 100%isopropanol comparing with the silk spun by replacing 2Rep-chromoprotein with chromoprotein only or NT-2Rep-chromoprotein. It’s also the most transmittance The stable feature of this particular silk spun supports our assumption to let the 2Rep region bounded to the chromoprotein assist the NT-2Rep-CT extensive region’s transition into beta-sheet formation as the 2Rep regions can lie parallel to each other and be formed into a beta-sheet structure cohesively without disruption to the dimer forming at NT or the amyloid-like fibril forming at CT when carrying out artificial silk spinning at low pH or in 100% isopropanol.
Due to the easily self-assemble property of amilCP chromoprotein, we were unable to artificially spin its mix with NT-2Rep-CT spidroin into continuous silk, become the syringe head was stuck immediately after the dose of mixture enters the syringe and amilCP starts to self-assemble and subside to the needle head.
Scanning electron microscopy of fibers
Aiming to verify the distribution uniformity of the mixture of recombinant chromoprotein and spidroin after spinning into fiber, we looked at our artificial spun silks under electron microscope. AmilCP is not scanned due to its lack of fluorescence. We randomly selected the cross section in our continuous silk, it has clearly shown that the fiber circularity is qualified as a uniformly distributed silk. For both sfGFP and eforRed, when 2Rep is added to the front of chromoprotein, the silk formed is has the smallest diameter among all the silks in the chromoprotein same group.