Difference between revisions of "Part:BBa K4614112"
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__TOC__ | __TOC__ | ||
==Cultivation, Purification and SDS-PAGE== | ==Cultivation, Purification and SDS-PAGE== | ||
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===Shaking Flask Cultivations=== | ===Shaking Flask Cultivations=== | ||
| + | ==Activity Analysis of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K863005 ECOL]== | ||
| + | <p align="justify"> | ||
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| + | Cross-linked engineering bacteria antibiotic filamentation experiment | ||
| + | Based on the cross-linking group of engineered bacteria ABCD, the specific information is shown in Table 1, we conducted the antibiotic filamentation experiment and tried to construct a new gene circuit to realize the cross-linking and silk binding at the molecular level. | ||
| + |  | ||
| + | Table. 1 Plasmids and genes induced into engineering bacteria.</center> | ||
| + | |||
| + | Based on literature review, we set up the following experimental group to use ampicillin to filamentation bacteria and simulate the possible cross-linking situation of filamentation bacteria after the expression of SulA gene to verify our conjusion: | ||
| + | |||
| + |  | ||
| + | Table. 2 The process and group setup of cross-linked engineering bacteria antibiotic filamentation experiment</center> | ||
| + | |||
| + | Laser microscope observation results are shown in Fig 1. | ||
| + | |||
| + |  | ||
| + | Fig. 1 The filamentated SpyCather could still be cross-linked to SpyTag under 1000 × fluorescence microscope. It is obvious that the degree of red and green fluorescence overlap in the experimental group (SpyTag+SpyCatcher) is greater than that in the other three control groups. | ||
| + | |||
| + | In microscopic observations, we found that a lot of green fluorescence binds to the poles of the red coryneform, which means that Wza can indeed position SpyCatcher at the poles after bacterial filaments. Even more surprising, we observed that some bacteria crosslinked to form a special linear, square and lotus structure, which means that when our engineered bacteria express both crosslinking and filamentation modules at the same time, it is very likely to form a micro-structure with special properties. | ||
| + | |||
| + |  | ||
| + | Fig. 2 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - linear | ||
| + | |||
| + |  | ||
| + | Fig. 3 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - close to square shape | ||
| + | |||
| + |  | ||
| + | Fig. 4 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - lotus shape | ||
| + | |||
| + | We were extremely excited to get these results less than 24 hours before the Wiki freeze. These results demonstrate that there is a high probability that the materials formed by our engineered bacteria will have special microstructure and therefore special properties. | ||
Revision as of 18:12, 11 October 2023
_ Encoding sequence of the fusion protein of Wza and SpyCatcher
Wza is an integral outer membrane lipoprotein, which is essential for group 1 capsule export in Escherichia coli. The transmembrane region is a novel α-helical barrel. Mature Wza, the best studied OMA member, is a 359-residue lipoprotein protein that forms SDS-stable octamers. The C terminus of each monomer is exposed on the cell surface, placing the acylated N terminus at the inner leaflet of the outer membrane, consistent with typical outer membrane lipoproteins[1]. The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag[2]. [1]Dong C, Beis K, Nesper J, et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein[J]. Nature, 2006,444(7116):226-229. [2]Hatlem, D.; Trunk, T.; Linke, D.; Leo, J.C. Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. Int. J. Mol. Sci. 2019, 20, 2129.
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 661
Profile
Name:Fusion protein Wza-SpyCatcher
Base Pairs: 1503 bp
Origin: Escherichia coli K12 & Streptococcus pyogenes, synthetic
Properties: Under certain conditions, Wza can display SpyCatcher，which can cross-link with SpyTag, specifically at the poles of the bacteria. cross-link with SpyTag.
Usage and Biology
The SpyTag-SpyCatcher system is widely usedLigation of targeting-antibody with antigen provided a simple route to vaccine generation. SpyRings, from head-to-tail cyclisation, gave major enhancements in enzyme resilience. Linking multiple SpyCatchers gave dendrimers for T-cell activation or Spy networks forming hydrogels for stem cell culture. Synthetic biology applications include integrating amyloid biomaterials with living bacteria, for irreversible derivatisation of biofilms with enzymes or nanoparticles. We also discuss further opportunities to apply and enhance SpyTag/SpyCatcher technology[3] We try to develop Wza as a new carrier protein, and use its characteristic of being relatively fixed on the cell wall to achieve our special requirements for the spatial location of the display protein. [3]Samuel C Reddington, Mark Howarth,Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher,Current Opinion in Chemical Biology,Volume 29,2015,Pages 94-99,ISSN 1367-5931,https://doi.org/10.1016/j.cbpa.2015.10.002.
Contents
Cultivation, Purification and SDS-PAGE
Shaking Flask Cultivations
Activity Analysis of ECOL
Cross-linked engineering bacteria antibiotic filamentation experiment
Based on the cross-linking group of engineered bacteria ABCD, the specific information is shown in Table 1, we conducted the antibiotic filamentation experiment and tried to construct a new gene circuit to realize the cross-linking and silk binding at the molecular level.
)
Table. 1 Plasmids and genes induced into engineering bacteria.</center>
Based on literature review, we set up the following experimental group to use ampicillin to filamentation bacteria and simulate the possible cross-linking situation of filamentation bacteria after the expression of SulA gene to verify our conjusion:
)
Table. 2 The process and group setup of cross-linked engineering bacteria antibiotic filamentation experiment</center>
Laser microscope observation results are shown in Fig 1.
)
Fig. 1 The filamentated SpyCather could still be cross-linked to SpyTag under 1000 × fluorescence microscope. It is obvious that the degree of red and green fluorescence overlap in the experimental group (SpyTag+SpyCatcher) is greater than that in the other three control groups.
In microscopic observations, we found that a lot of green fluorescence binds to the poles of the red coryneform, which means that Wza can indeed position SpyCatcher at the poles after bacterial filaments. Even more surprising, we observed that some bacteria crosslinked to form a special linear, square and lotus structure, which means that when our engineered bacteria express both crosslinking and filamentation modules at the same time, it is very likely to form a micro-structure with special properties.
)
Fig. 2 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - linear
)
Fig. 3 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - close to square shape
)
Fig. 4 The engineered bacteria expressing SpyTag (green) and SpyCatcher (red), respectively, cross-link to form a special structure - lotus shape
We were extremely excited to get these results less than 24 hours before the Wiki freeze. These results demonstrate that there is a high probability that the materials formed by our engineered bacteria will have special microstructure and therefore special properties.