Difference between revisions of "Part:BBa K3122002"
| Line 3: | Line 3: | ||
<partinfo>BBa_K3122002 short</partinfo> | <partinfo>BBa_K3122002 short</partinfo> | ||
| − | . | + | This composite part consists of our AEGIS's lamB display system for Gram- bacteria, expressing a 6 histidine tag. This part was used as base of the cell purification system for the SELEX procedure. Histidines may bind to nickel magnetic beads, allowing the separation and retention of the cells under a magnetic field. |
| + | |||
| + | To see the design of this part go to the simple parts pages. | ||
| + | |||
| + | <h2> Biology </h2> | ||
| + | |||
| + | LamB is maltoporin precursor of Gram-negative bacteria. This outer membrane proteins family is involved in transport of maltodextrins across the outer membrane. Besides, LamB is the receptor of several different bacteriophagues as lambda fague. | ||
| + | Gene sequence of LamB [Escherichia coli str. K-12 substr. MG1655] was obtained from EcoGene:EG10528. | ||
| + | Histidine tag is derived from BBa_K1223006, which was designed using E. coli codon usage tables. | ||
| + | |||
| + | <h2> Characterization </h2> | ||
| + | |||
| + | Characterization of this part has been conducted by building a transcriptional unit BBa_K3122004 and performing assays with nickel beads in order to check the interaction between them and its force. This transcriptional unit was assembled in pARK1 alpha plasmid including the following parts: | ||
| + | |||
| + | <ul> | ||
| + | <li><partinfo>BBa_K3122003</partinfo>: Promoter J23107 adapted to type IIS assembly</li> | ||
| + | <li><partinfo>BBa_K2656009</partinfo>: RBS B0030 adapted to type IIS assembly</li> | ||
| + | <li><partinfo>BBa_K3122002</partinfo>: coding sequence. This composite part is built with BBa_K3122000 (LamB protein) and BBa_K3122001 (6xHis tag) creating our AEGIS' lamB display system exposing 6xHis Tag.</li> | ||
| + | <li><partinfo>BBa_K2656026</partinfo>: Terminator B0015 adapted to type IIS assembly</li> | ||
| + | </ul> | ||
| + | |||
| + | <h2> How to use it </h2> | ||
| + | |||
| + | This is an example of how this new display system that we created can be used by future iGEM teams on their MoClo reactions with the desired tag to create level 1 constructs. The only requirement is to adapt the tag sequence to the new scars designed. | ||
| + | |||
| + | <h2> References </h2> | ||
| + | |||
| + | C. Sousa, A. Cebolla and V. de Lorenzo, "Enhanced metalloadsorption of bacterial cells displaying poly-His peptides", Nature Biotechnology, vol. 14, no. 8, pp. 1017-1020, 1996. Available: 10.1038/nbt0896-1017 [Accessed 20 October 2019]. | ||
| + | |||
| + | L. Sciences, P. Biology, P. Center, P. Library, P. Methods and H. Purification, "His-tagged Proteins–Production and Purification | Thermo Fisher Scientific - UK", Thermofisher.com, 2019. [Online]. Available: https://www.thermofisher.com/es/es/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html. [Accessed: 20- Oct- 2019]. | ||
| + | |||
| + | J. Kim, C. Valencia, R. Liu and W. Lin, "Highly-Efficient Purification of Native Polyhistidine-Tagged Proteins by Multivalent NTA-Modified Magnetic Nanoparticles", Bioconjugate Chemistry, vol. 18, no. 2, pp. 333-341, 2007. Available: 10.1021/bc060195l. | ||
| + | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
<partinfo>BBa_K3122002 parameters</partinfo> | <partinfo>BBa_K3122002 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
Latest revision as of 02:59, 22 October 2019
AEGIS' lamB display system exposing 6xHis Tag
This composite part consists of our AEGIS's lamB display system for Gram- bacteria, expressing a 6 histidine tag. This part was used as base of the cell purification system for the SELEX procedure. Histidines may bind to nickel magnetic beads, allowing the separation and retention of the cells under a magnetic field.
To see the design of this part go to the simple parts pages.
Biology
LamB is maltoporin precursor of Gram-negative bacteria. This outer membrane proteins family is involved in transport of maltodextrins across the outer membrane. Besides, LamB is the receptor of several different bacteriophagues as lambda fague. Gene sequence of LamB [Escherichia coli str. K-12 substr. MG1655] was obtained from EcoGene:EG10528. Histidine tag is derived from BBa_K1223006, which was designed using E. coli codon usage tables.
Characterization
Characterization of this part has been conducted by building a transcriptional unit BBa_K3122004 and performing assays with nickel beads in order to check the interaction between them and its force. This transcriptional unit was assembled in pARK1 alpha plasmid including the following parts:
- BBa_K3122003: Promoter J23107 adapted to type IIS assembly
- BBa_K2656009: RBS B0030 adapted to type IIS assembly
- BBa_K3122002: coding sequence. This composite part is built with BBa_K3122000 (LamB protein) and BBa_K3122001 (6xHis tag) creating our AEGIS' lamB display system exposing 6xHis Tag.
- BBa_K2656026: Terminator B0015 adapted to type IIS assembly
How to use it
This is an example of how this new display system that we created can be used by future iGEM teams on their MoClo reactions with the desired tag to create level 1 constructs. The only requirement is to adapt the tag sequence to the new scars designed.
References
C. Sousa, A. Cebolla and V. de Lorenzo, "Enhanced metalloadsorption of bacterial cells displaying poly-His peptides", Nature Biotechnology, vol. 14, no. 8, pp. 1017-1020, 1996. Available: 10.1038/nbt0896-1017 [Accessed 20 October 2019].
L. Sciences, P. Biology, P. Center, P. Library, P. Methods and H. Purification, "His-tagged Proteins–Production and Purification | Thermo Fisher Scientific - UK", Thermofisher.com, 2019. [Online]. Available: https://www.thermofisher.com/es/es/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/his-tagged-proteins-production-purification.html. [Accessed: 20- Oct- 2019].
J. Kim, C. Valencia, R. Liu and W. Lin, "Highly-Efficient Purification of Native Polyhistidine-Tagged Proteins by Multivalent NTA-Modified Magnetic Nanoparticles", Bioconjugate Chemistry, vol. 18, no. 2, pp. 333-341, 2007. Available: 10.1021/bc060195l.