Difference between revisions of "Part:BBa K3122001"
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__NOTOC__ | __NOTOC__ | ||
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<partinfo>BBa_K3122001 short</partinfo> | <partinfo>BBa_K3122001 short</partinfo> | ||
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| − | < | + | This part is derived from BBa_K1223006. It contains 6 histidine residues, and it has been adapted to AEGIS' lamB display system in order to be used as the heterologous peptide tag expressed. |
| − | == | + | |
| + | To see the design of this part go to the Design tab. | ||
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| + | <h2> Biology </h2> | ||
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| + | 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. | ||
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| + | <h2> Characterization </h2> | ||
| + | |||
| + | ==Characterization of Histidine-based separation== | ||
| + | Histidines are used as base of the separation system of our Robo-SELEX protocol. Several alternative strategies were designed, all based on the same concept: the LamB - 6xHis tag functions as an anchoring system, so the cell is retained while aptamers are eluted. | ||
| + | |||
| + | <ul> | ||
| + | <li>First strategy: magnetic resin. | ||
| + | </li> | ||
| + | <li>Second strategy: magnetic beads. Following the principle explained above, we tried the same protocol with a different material, this time using cobalt magnetic beads to bind to the Histidine Tag, expressed throughout the surface of E. cholira (J. Kim, 2007). | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | ==Results== | ||
| + | |||
| + | <ol type="1"> | ||
| + | <li>. We conducted a first assay with the magnetic resin using the maximum values for all the variables we wanted to optimize (initial concentration of cells and incubation time), and as the proportion of cells found after plating the dilutions we were unable to drag or retain enough cells. | ||
| + | |||
| + | After analyzing our results, we concluded that due to the small size of the particles in the resin, they would be too compact with a pore size too narrow to allow let the cell access to the inside of the resin matrix. | ||
| + | </li> | ||
| + | <li> | ||
| + | We attempted to separate the cells with the cobalt magnetic beads. We thought that the individuality of the beads, which are present in the solution without aggregating in a small-pore-sized resin, would correct those problems. An assay was conducted as a proof of concept, comparing the absorbance between Eppendorfs that suffer E. cholira separation with and without expressing the Histidine tag. So, after performing the assay in the OT2, we measured absorbance at 640nm of the resulting 96 well-plate. | ||
| + | |||
| + | The results obtained in this assay showed a significant difference between the control cells pop6510 harbouring vector pARK1-LamB (without expressing the anchor systems) and the pop6510 harbouring pARK1LamB-6xHis. With this, we confirmed that there was an interaction between the Tag and the cobalt beads. After analyzing the results obtained in the assay we saw that the efficiency of the process was around 10%, which was not enough to conduct a proper SELEX protocol due to the number of aptamers in each SELEX round that we would lose in the remaining 90% of the cells. | ||
| + | </li> | ||
| + | </ul> | ||
| + | |||
| + | <h2> How to use it </h2> | ||
| + | |||
| + | AEGIS' lamB 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, as this part has been adapted. | ||
| − | + | <partinfo>BBa_K SequenceAndFeatures</partinfo> | |
| − | + | ||
| − | <partinfo> | + | |
| + | <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]. | |
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Revision as of 22:35, 21 October 2019
6xHis Tag (Adapted to AEGIS' lamB display system)
This part is derived from BBa_K1223006. It contains 6 histidine residues, and it has been adapted to AEGIS' lamB display system in order to be used as the heterologous peptide tag expressed.
To see the design of this part go to the Design tab.
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.
Characterization
Characterization of Histidine-based separation
Histidines are used as base of the separation system of our Robo-SELEX protocol. Several alternative strategies were designed, all based on the same concept: the LamB - 6xHis tag functions as an anchoring system, so the cell is retained while aptamers are eluted.
- First strategy: magnetic resin.
- Second strategy: magnetic beads. Following the principle explained above, we tried the same protocol with a different material, this time using cobalt magnetic beads to bind to the Histidine Tag, expressed throughout the surface of E. cholira (J. Kim, 2007).
Results
- . We conducted a first assay with the magnetic resin using the maximum values for all the variables we wanted to optimize (initial concentration of cells and incubation time), and as the proportion of cells found after plating the dilutions we were unable to drag or retain enough cells. After analyzing our results, we concluded that due to the small size of the particles in the resin, they would be too compact with a pore size too narrow to allow let the cell access to the inside of the resin matrix.
- We attempted to separate the cells with the cobalt magnetic beads. We thought that the individuality of the beads, which are present in the solution without aggregating in a small-pore-sized resin, would correct those problems. An assay was conducted as a proof of concept, comparing the absorbance between Eppendorfs that suffer E. cholira separation with and without expressing the Histidine tag. So, after performing the assay in the OT2, we measured absorbance at 640nm of the resulting 96 well-plate. The results obtained in this assay showed a significant difference between the control cells pop6510 harbouring vector pARK1-LamB (without expressing the anchor systems) and the pop6510 harbouring pARK1LamB-6xHis. With this, we confirmed that there was an interaction between the Tag and the cobalt beads. After analyzing the results obtained in the assay we saw that the efficiency of the process was around 10%, which was not enough to conduct a proper SELEX protocol due to the number of aptamers in each SELEX round that we would lose in the remaining 90% of the cells.
</ul>
How to use it
AEGIS' lamB 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, as this part has been adapted.
No part name specified with partinfo tag.