Part:BBa_K343004

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.

Datasheet for K343004



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For further details on characterization see experience

Part background

More than 80% of all known bacterial species express flagella [1]. The synthesis of the flagella is a complex and energy consuming process, so the expression of the involved proteins is tightly regulated by the extracellular environment. One of the most well studied flagella synthesis systems is that of Eschericia coli. Here the expression of the master regulator FlhDC operon is controlled i.e. by pH, salt concentration or the availability of nutrients. Apart from initiation of flagella synthesis FlhDC is also a repressor of cell metabolism as cell growth and flagellation does not occur at the same time. FlhDC is a hexameric transcription factor that consists of four FlhD subunits and two FlhC subunits (FlhD₄C₂). It constitutes the first class in the three classed flagella synthesis cascade. Class II consist of genes encoding proteins that make up the basal body, as well as the alternative transcription factor, σ²⁸, which is responsible for the expression of the class III genes. These genes in turn encodes the proteins that composes the tail filaments.

Studies of insertion elements (IS) upstream of the flhDC operon, has shown that an upregulation of the expression of the operon is responsible for the motility of the E. coli MG1655 strain. [2]It has also been shown that overexpression of the FlhDC operon restores motility in mutants that have been made immotile [3].

Usage and parameters

Performance

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 strain MG1655.
Plasmids: PSB1C3 (high-copy), PSB3K3 (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 [4]. NpHtrII is thought to function in signal transduction and activation of microbial signalling cascades [5].

A single article has been written about haloarchaea in humans indicating that these played a role in patients with inflammatory bowel disease [6], 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.

Results

Our results from the motility assay with semi-solid agar confirm that the biobrick does in fact increase the motility of the cells. We believe this is caused by hyperflagellation facilitated by the overexpression of the flhD/C operon.
The stability of pSB1C3-K343004 is most likely <20 generations, however the stability of pSB3T5-K343004 was not determined.
The growth assay showed no significant difference between the wild type and the cells containing either pSB1C3-K343004 or pSB3K3-K343004.
In a phase contrast microscope we observed that Mg1655 containing pSB1C3-K343004 was longer that wild type. This support the theory that the cells are hyperflagellated and therefore stays longer in the G2-phase in the cell cycle. For further information, raw data and background of the assay see [http://2010.igem.org/Team:SDU-Denmark/K343004 characterization of K343004] on our team wiki.

References

1. O. Soutourina , PN. Bertin [http://www.ncbi.nlm.nih.gov/pubmed/14550943 Regulation cascade of flagellar expression in Gram-negative bacteria.]
   
2. O. Soutourina, A. Kolb, E. Krin, C. Laurent-Winter, S. Rimsky, A. Danchin, and P. Bertin[http://jb.asm.org/cgi/content/short/181/24/7500 Multiple Control of Flagellum Biosynthesis in Escherichia coli: Role of H-NS Protein and the Cyclic AMP-Catabolite Activator Protein Complex in Transcription of the flhDC Master Operon]
   
3. Eric J. Gauger, Mary P. Leatham, Regino Mercado-Lubo, David C. Laux, Tyrrell Conway, and Paul S. Cohen [http://iai.asm.org/cgi/content/abstract/75/7/3315 Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine{triangledown}]
   
4. Jung KH, Spudich EN, Trivedi VD, Spudich JL. [http://www.ncbi.nlm.nih.gov/pubmed An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli]. J. Bacteriol. 2001 Nov;183(21):6365-6371.
5. 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.
6. Oxley APA, Lanfranconi MP, Würdemann D, Ott S, Schreiber S, McGenity TJ, et al. [http://www.ncbi.nlm.nih.gov/pubmed Halophilic archaea in the human intestinal mucosa]. Environ Microbiol [Internet]. 2010 Apr 23 [cited 2010 Oct 26].

Sequence and Features

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 689
  
• 1000
  COMPATIBLE WITH RFC[1000]