Difference between revisions of "Part:BBa K3122000"
 
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<h2> Characterization </h2>
 
<h2> Characterization </h2>
  
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[[File:T--MADRID_UCM--petridish.png|250px|thumb|centro|alt=fage analysis.|Fague assay 1 and 2 results: 1) MG1655 positive control, 2) Pop6510 - LamB, 3) Pop6510 - LamB - 6xHis, 4) Pop6510 negative control.]]
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Characterization of this part has been conducted by building a transcriptional unit <partinfo>BBa_K3122005</partinfo> and performing Lambda fague assays. This transcriptional unit was assembled in pARK1 alpha plasmid including the following parts:
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For this characterization, we chose to perform a rather simple test that nonetheless gives conclusive results: the Lambda phage assay.
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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.
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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.
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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:
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<ul>
 
<ul>
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<li>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. </li>
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<li><partinfo>BBa_K3122003</partinfo>: Promoter J23107 adapted to type IIS assembly</li>
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<li>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.</li>
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<li><partinfo>BBa_K2656009</partinfo>: RBS  B0030 adapted to type IIS assembly</li>
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<li><partinfo>BBa_K3122000</partinfo>: LamB coding sequence. This part is our AEGIS' lamB display system.</li>
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<li><partinfo>BBa_K2656026</partinfo>: Terminator B0015 adapted to type IIS assembly</li>
 
</ul>
 
</ul>
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We used regular MG1655 E. coli cells (expressing LamB normally) as positive control, and our pop6510 cells as negative control.
 
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The results obtained were positive:
 
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<ul>
 
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<li>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.
 
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</li>
 
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<li>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.
 
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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.
 
  
  
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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.
 
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.
  
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<partinfo>BBa_K SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3122000 SequenceAndFeatures</partinfo>
  
 
<h2>References</h2>
 
<h2>References</h2>
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<h2>Judging</h2>
 
<h2>Judging</h2>
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The advantages of this approach are notable: not only can it be used for bacteria with cell wall ensuring its right functioning, but also it is really simple and easy to use following MoClo standard.
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The sharp design of this part allows a perfect scarless ligation of the desired tag into the permissive loop of LamB, type IIS adapted sequence, in the same one-pot reaction where MoClo is ocurring. It guarantees perfect expression of the tag.

Latest revision as of 00:47, 22 October 2019


AEGIS's lamB display system for Gram- bacteria


E. coli LamB is one of the best characterized and most flexible heterologous peptide carriers. This is due to its extracelular domains, consisting mainly of long loops. One of these loops can tolerate insertions of rather long sequences, between amino acid residues 153 and 154 (Sousa, 1996).

Our group have adapted this functional display system to type IIS enzymes grammar, and called it AEGIS's lamB display system for Gram-negative bacteria. This part was designed to use the outer membrane protein lamB of E. coli K12 strain to express any tag or short sequence to the extracellular medium on its specific permissive loop. The advantages of this approach are notable: not only can it be used for bacteria with cell wall ensuring its right functioning, but also it is really simple and easy to use following MoClo standard.

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 this part has been conducted by building a transcriptional unit BBa_K3122005 and performing Lambda fague assays. 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_K3122000: LamB coding sequence. This part is our AEGIS' lamB display system.
  • BBa_K2656026: Terminator B0015 adapted to type IIS assembly


How to use it

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.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 152
    Illegal AgeI site found at 464
    Illegal AgeI site found at 1229
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 467
    Illegal BsaI.rc site found at 461

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].

Judging

The sharp design of this part allows a perfect scarless ligation of the desired tag into the permissive loop of LamB, type IIS adapted sequence, in the same one-pot reaction where MoClo is ocurring. It guarantees perfect expression of the tag.