Juiceyprint Proposal

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London Biohackers DRAFT IGEM Research Proposal "JuiceyPrint" (working title)

Background and motivation

Layering of macromolecular substrates has many potential applications in biotechnology, biomedical science and other technology projects and as such there is a strong motivation for constructing sheets of biopolymers including cellulose, collagen and silk with micron scale architecture. Inspiration here is drawn from lithographic methods where differential light exposure on a receptive surface can be used to control the deposition or removal of a substance of choice. In this case it is intended to be mediated by a strain of light sensitive bacterium whose metabolic production of a selected structural macromolecule can be regulated through its coupling to a light sensitive signal transduction pathway.

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previous similar work

Levskaya et al.(2005) "engineered E.Coli to see light".

Engineering Escherichia coli to see light

Abstract:

These smart bacteria 'photograph' a light pattern as a high-definition chemical image. We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to 'print' complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.

Deng et al.(2013)

Identification and characterization of non-cellulose-producing mutants of Gluconacetobacter hansenii generated by Tn5 transposon mutagenesis

Abstract:

The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel(-)) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript "Ax" indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel(-) mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel(-) mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding β-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis.


Random iGem projects

2008:Imperial

2010:Osaka

2012:NYU:Gallatin

2013:Biwako_Nagahama

Working hypothesis and questions

how we expect things to work, potential shortcomings and how to get round them, specific problems/outcomes we hope to address

(1) Obtain non-cellulose-producing G.hansenii (which one?) or do the knockout ourselves.

(2) do another knockout (what? why?)

(3) add our plasmid with a light activated promoter and replacement for the gene knocked out in (1)

(4) juicyprint!


Experimental design and methodology

details of genes, components/biobricks etc and procedures.

Future prospects

if this works what might it lead to, what can we look at next or refine

Bibliography

Levskaya, A., Chevalier, A. A., Tabor, J. J., Simpson, Z. B., Lavery, L. A., Levy, M., Davidson, E. A., Scouras, A., Ellington, A. D., Marcotte, E. M. and Voigt, C. A. (2005) ‘Synthetic biology: Engineering Escherichia coli to see light’, Nature, 438(7067), pp. 441–442.

PDF via google scholar

PDF from mailing list archive

@article{levskaya2005synthetic,
  title={Synthetic biology: engineering Escherichia coli to see light},
  author={Levskaya, Anselm and Chevalier, Aaron A and Tabor, Jeffrey J and Simpson, Zachary Booth and Lavery, Laura A and Levy, Matthew and Davidson, Eric A and Scouras, Alexander and Ellington, Andrew D and Marcotte, Edward M and others},
  journal={Nature},
  volume={438},
  number={7067},
  pages={441--442},
  year={2005},
  publisher={Nature Publishing Group}
}


Deng, Y., Nagachar, N., Xiao, C., Tien, M. and Kao, T. (2013) ‘Identification and characterization of non-cellulose-producing mutants of Gluconacetobacter hansenii generated by Tn5 transposon mutagenesis’, Journal of bacteriology, 195(22), pp. 5072–5083.

full text html from Journal of Bacteriology

PDF from archive of mailing list

G.Hansenii ATCC 23769 whole genome sequence