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Part:BBa_K343004

Designed by: Louise Linnebjerg Bohn Christoffersen, Pernille Marie Madsen, Sheila Maibom-Thomsen   Group: iGEM10_SDU-Denmark   (2010-07-05)
Revision as of 20:08, 27 October 2010 by Toand (Talk | contribs) (→‎References)

Flagella overekspression


This part entails a TetR repressed POPS/RIPS generator (including RBS), the FlhDC master operon and a dual-terminator. This part will cause hyperflagellated cells.

Part background


The flagella regulon in E. coli is composed of at least 50 genes organized in no less than 14 operons that all contribute to the synthesis and operation of flagella. The operons are synthesized in a three-level transcriptional cascade where the FlhDC operon is the master regulator at the top of the cascade. The flagella regulon is tightly controlled by nutritional and environmental conditions, E. coli starved of amino acids showed temporarily decrease of the flagella regulon transcripts which are needed for the synthesis and operation of the flagellum.[http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06939.x/full (1)] The synthesis and assembly of flagella are regulated by the transcriptional cascade composed of three levels of gene products (class I, -II and –III). Class I genes consist of a single operon encoding the proteins FlhD and FlhC that form a multimeric (FlhD4C2) transcriptional activation complex. This ‘master regulator’ stimulates transcription by binding upstream of Class II promoters. Class II genes encode proteins that assemble to form the basal body and hook of the flagellum, as well as the fliA gene that encodes the alternative σ factor σ28, also called σF. σ28 binds to RNA polymerase (RNAP) core enzyme and directs it to Class III promoters. Class III genes encode the rest of the structural genes of the flagellum, including fliC encoding flagellin, as well as the chemotaxis apparatus. [http://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2009.06939.x/full (1)]
It has been shown that overexpression of the FlhDC operon restores motility in mutants that have been made immotile [http://jb.asm.org/cgi/content/short/181/24/7500 (2)]. Also, overexpression of FlhDC in the E. coli K12 strain MG1655 made the cells hypermotile.[http://iai.asm.org/cgi/content/abstract/75/7/3315 (3)]

Usage and parameters

Usage

Performance

Response time:

Production rate:

Plasmid stability: A stability assay have been preformed the data can be accessed [http://2010.igem.org/Team:SDU-Denmark/project-p#Stability_assay_2 here].
Almost all of the bacteria had shedded the plasmid after 20 generations, suggesting that the plasmid is only stable within the cell for a few generations (<20). This is presumably due to the strain brought upon the bacteria by the plasmid. Thereby when the bacteria are carrying a high-copy plasmid like pSB1C3-K343004 it is to expect that the bacteria will quickly shed the plasmid when no longer exposed to a selection pressure. It is likely to believe that pSB3C5-K343004, since being a low-copy plasmid, will not excert as much strain on the bacteria, and might therefore be stable for more genrations than pSB1C3-K343004. Therefore a stability assay of this plasmid might be of interest.

Growth rate:A growth assay have been preformed the data can be accessed [http://2010.igem.org/Team:SDU-Denmark/project-p#Growth_assay_2 here]
From our data we see no significant difference between the plasmid carrying bacteria and the wild type. This can be said to be quite contradictory to our results obtained from the stability assay. The transitory stability of pSB1C3-K343004 suggests that it is highly unfavourable for the bacteria, wherefore it might be expected that the growth of the bacteria congaing this plasmid would be affected. Thus, however much a disadvantage the plasmid pose to the bacteria, their growth are not significantly influenced by the plasmid. The added reproduction load due to the plasmids, might also prolong the lag phase of the bacteria. Whether this is the case can not be concluded based on this experiment as no lag phase was observed in this experiment.

Compatibility

This brick has been tested in the following plasmids and stains:

Chassis: E. coli TOP10, E. coli MG1655.

Plasmids: PSB1C3 (high-copy), PSB3K5 (low-copy).


Risk-assessment

General use

This BioBrick poses no treat to the welfare of people working with it, as long as this is done in at least a level 1 safety lab by trained people. No special care is needed when working with this BioBrick.

Potential pathogenicity

This BioBrick consists of three different parts: The first 224 amino acid residues come from the NpSopII gene from Natronomonas pharaonis, encoding a blue-light photon receptor with 15 residues removed at the C-terminal. The following 9 amino acids are a linker. The last part is HtrII fused with Tar from E. coli. The complex' first 125 amino acid residues come from HtrII and the remaining 279 from Tar ([http://2010.igem.org/Team:SDU-Denmark/safety-b#References 7]). NpHtrII is thought to function in signal transduction and activation of microbial signalling cascades ([http://2010.igem.org/Team:SDU-Denmark/safety-b#References 8]).

A single article has been written about haloarchaea in humans indicating that these played a role in patients with inflammatory bowel disease ([http://2010.igem.org/Team:SDU-Denmark/safety-b#References 9]), but there is no evidence that the genes this BioBrick is made from or any near homologs are involved in any disease processes, toxic products or invasion properties. They do not regulate the immune system in any way.

Environmental impact

The BioBrick does not produce a product that is secreted into the environment, nor is it’s gene product itself toxic. It would not produce anything that distrupt natural occurring symbiosis.

The BioBrick might increase a bacteria’s ability to find nutrients and as such ease its ability to replicate and spread in certain dark environments. On the other hand the BioBrick is very large and this will naturally slow down its replication rate. Generally we do not believe this BioBrick will make its host able to outcompete natural occurring bacteria, simply because it’s function is not something that will give its host a functional advantage.

Possible malign use

This BioBrick will not increase its hosts ability to survive in storage conditions, to be aerosoled, to be vaporized or create spores. None of its proteins regulate or affect the immune system or are pathogenic towards humans and animals.

==Resources==

Datasheet for BioBrick.

PDB file for protein structure.

References

  1. Mennes N, Klare JP, Chizhov I, Seidel R, Schlesinger R, Engelhard M. [http://www.ncbi.nlm.nih.gov/pubmed Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli.] FEBS Lett. 2007 Apr 3;581(7):1487-1494.
  2. ENZYME entry 1.14.99.36 [Internet]. [cited 2010 Oct 13];Available from: http://www.expasy.org/cgi-bin/nicezyme.pl?1.14.99.36
  3. von Lintig J, Dreher A, Kiefer C, Wernet MF, Vogt K. Analysis of the blind Drosophila mutant ninaB identifies the gene encoding the key enzyme for vitamin A formation in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2001 Jan 30;98(3):1130 -1135.
  4. Retinal - Wikipedia, the free encyclopedia [Internet]. [cited 2010 Oct 13];Available from: http://en.wikipedia.org/wiki/Retinal
  5. Part:BBa K274210 - parts.igem.org [Internet]. [cited 2010 Oct 13];Available from: https://parts.igem.org/Part:BBa_K274210
  6. Bryant DA, Frigaard N. Prokaryotic photosynthesis and phototrophy illuminated. Trends Microbiol. 2006 Nov;14(11):488-496.
  7. Retinaldehyde - PubChem Public Chemical Database [Internet]. [cited 2010 Oct 13];Available from: http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1070
  8. ninaB neither inactivation nor afterpotential B [Drosophila melanogaster] - Gene result [Internet]. [cited 2010 Oct 13];Available from: http://www.ncbi.nlm.nih.gov/gene/41678
  9. von Lintig J, Vogt K. Filling the Gap in Vitamin A Research. Journal of Biological Chemistry. 2000 Apr 21;275(16):11915 -11920.
  10. ENZYME: 1.14.99.36 [Internet]. [cited 2010 Oct 13];Available from:http://www.genome.jp/dbget-bin/www_bget?ec:1.14.99.36
  11. Kelley LA & Sternberg MJE. Protein structure prediction on the web: a case study using the Phyre server. Nature Protocols. 4, 363 - 371 (2009).
  12. Spiegl N, Didichenko S, McCaffery P, Langen H, Dahinden CA. Human basophils activated by mast cell-derived IL-3 express retinaldehyde dehydrogenase-II and produce the immunoregulatory mediator retinoic acid. Blood. 2008 Nov 1;112(9):3762-71.
  13. Russell RM. The vitamin A spectrum: from deficiency to toxicity. American Journal of Clinical Nutrition, Vol. 71, No. 4, 878-884, April 2000.
  14. Pasquali D, Thaller C, Eichele G. Abnormal level of retinoic acid in prostate cancer tissues. J Clin Endocrinol Metab. 1996 Jun;81(6):2186-91.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 765
    Illegal BamHI site found at 500
    Illegal BamHI site found at 1757
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1361
    Illegal BsaI site found at 1809


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