Difference between revisions of "Part:BBa K1223006:Experience"

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Characterization of BBa_K1223006 by 2019 MADRID_UCM team

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

Background

This part was used as a 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.

We decided to express a six-unit histidine tag in our LamB display system, in the permissive loop of LamB . The peculiarity of this tag is that histidines bind strongly to nickel beads subjected to a magnetic field. Since histidine is a polar amino acid, negatively charged (due to its multiple amine groups) in a wide range of pH, it can easily interact with ions of metallic particles (Thermofisher, 2019). Using this approach, we suggest that by expressing enough histidine tags in the membrane of our target cells we would be able to induce conjugation between magnetic beads and cells when incubated together in the magnetic module. For further information, visit our Robo-SELEX page .

Besides, by using this promoter of medium strength, we ensure the expression of our protein without overburdening the cell membrane, and therefore, withput killing the cell.

Experiments

Fague assay 1 and 2 results: 1) MG1655 positive control, 2) Pop6510 - LamB, 3) Pop6510 - LamB - 6xHis, 4) Pop6510 negative control.

For this characterization, we chose to perform a rather simple test that nonetheless gives conclusive results: the Lambda phage assay. The Lambda phage is a bacteriophage which infects the cell using the LamB protein. We used pop6510 cell line, which does not express LamB. Therefore, if there is no expression of our constructs, the phage cannot infect the cell and there will not be any lysis. On the other hand, if the Lamb display system is expressed properly, the phage will be able to infect the cell, and lysis spots shall appear in the Petri dish.

In this experiment, we characterized the expression in the outer membrane of the LamB protein both with the 6xHis tag and without. We aimed to tell if we had achieved a correct expression of the protein, and if the addition of the His-tag into the permissive loop would compromise this expression.

To prepare the test, we first dabbed the white colonies and made a liquid inoculum. The grown inoculum was treated as explained in the Fague lysis protocol, and then we conducted two different assays:

• Plating in LB agar plates a mix of soft-agar and the result of an incubation of 100μl of the liquid inoculum with 100 μl of phage.
• Plating soft-agar with the bacteria inoculum in LB agar plates with known droplets of Lambda phage: 5,6 and 7 μl in three punctual locations in the Petri dish.

We used regular MG1655 E. coli cells (expressing LamB normally) as positive control, and our pop6510 cells as negative control. The results obtained were positive:

• In the first assay, negative control plate was covered with a bed of cells and positive control plate was clean of cells, as expected. About problem samples, we could observe very few colonies in the petri dish, meaning that the majority of the cells were expressing LamB and therefore lysis has occurred in a high percentage of cells. In the second assay, similar good results were obtained: bald patches appeared in the zone where the Lambda phage was inoculated in the case of problem samples and positive control, while negative control had grown generously.
• In conclusion, the assays could be confirmed as successful. The results explained above were very similar in both constructions, confirming that the introduction of the Tag does not affect the correct expression of LamB in the E. coli outer membrane. However, this result should not be extrapolated to every tag. Bigger or longer tags may lead to misleading results, making this assay unuseful to check its presence.

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

1. 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.
2. 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.
   

   Figure 1:Histidine Tag cell separation using the second approach explained

   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.

References

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.

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Characterization of BBa_K1223006 by 2022 Thailand_RIS team

Background

Similarly to what the 2019 Madrid team proposed, this part holds the potential to be used in other ways of separation. For our experiment, the Histidine tag was used as a part of purification through His column. Since Histidine contains polar properties, metal ions such as Ni2+ or Cu2+, will attract the histidine and allow for bonds to form. This attraction will form strong bonds that will cause the His-tags to form bonds with the linings on the side of the column, allowing for the washing of surplus proteins, without losing our target protein.

Experiments

For our experiment, we had to undergo sonication to remove our protein from the host cell itself. However, when sonication occurs, even though the host cell is destroyed and proteins are free, all of the proteins are merged together. Thus, we had to separate our target protein from the rest of the proteins that kept the cells functioning. To accomplish this, we had to depend upon the His-tags. Within the amino acids of our peptide sequence Histidine which contains polar properties. With this, we decided to use His-columns, columns that are lined on the edges with metal ions, to separate our target protein from the rest. The histidine within our protein sequence will be attracted to the metal ions on the sides of the column due to the bonds that will be formed between the Histidine and metal ions. Since the other protein doesn’t contain Histidine in its sequences, no bonding will occur which will cause it to continually float in the solution mixture, separating it from our target protein. As we wash away the solution mixture, none of our target protein will be removed since it's still bonded to the sides. Afterwards, we wash the column with imidazole, eluting the protein. The imidazole is capable of eluting the protein since it competes with the histidine to bind with the metal ions lining the his columns.

SDS-page after sonication of host cells. Targeted protein is shown as the highlighted bands. Other bands are different proteins that were present in host cells after Sonication.

Results

After eluting the column with imidazole, we ran the collected protein on gel electrophoresis. We determined if the protein is correct through the band sizes that are present. Our peptide, Bombolitin, requires band sizes of 125 bp.

This shows that the protein that was collected was the correct one, with the base pairs of the bombolitin being between 100 and 200 base pairs.

References

“His-Tag Purification.” Bio, https://www.bio-rad.com/featured/en/his-tag-purification.html.