Difference between revisions of "Part:BBa K274001"

Revision as of 03:32, 20 October 2016

melanin pigment

Produces dark brown pigment output. The gene (melA) codes for a tyrosinase which produces the pigment from L-tyrosine. Production of the pigment requires the addition of copper and L-tyrosine supplements (the copper acts as a cofactor for the gene product) but no other precursors. The BioBrick sequence includes the native ribosome binding site.

iGEM Concordia 2016 is currently characterizing this part in more efficient manner by improving the expression of melA through a point mutation. Refer to parts BBa_K2045004 and BBa_K2045005.

Characterization by iGEM Concordia 2016

iGEM Concordia 2016 aimed to synthesize nanoparticles for the overall goal of equipping cells with "nano-weapons" and having them fight in a microfluidics chip, in our project titled "Combat Cells: League of Enhanced MicroGladiators." One of our methods of nanoparticle synthesis and attachment to cells is titled the recombinant method. Through the use of the protein MelA with its substrate L-DOPA, eumelanin is formed, which forms gold nanoparticles from gold ions in solution.

IGEM Concordia 2016 aimed to improve the characterization of MelA by determining MelA's ability to use tyrosine as a substrate in place of L-DOPA. We cloned MelA's sequence into a plasmid, with MelA expression under the control of a constitutive promoter. This would allow us to determine if MelA had any cytotoxic effects.

After transforming this plasmid into DH5alpha E. coli cells, we set up an experiment to test MelA's ability to use tyrosine as a substrate. We set up untransformed DH5alpha cells and just tyrosine wells as controls, alongside MelA-transformed DH5Alpha cells, all containing 2mM L-tyrosine. The wells contained 180 microlitres of cells at a starting OD600 of 0.5. We placed this plate into a Tecan-Sunrise, which measured the OD450 of the well contents at every 20 minutes for 2.5 days. The value of OD450 is directly related to the amount of melanin being formed in the wells. The following graph represents our obtained data.

Graphed for each of the wells containing DH5alpha cells (transformed with MelA and not transformed) is the average of three replicates. The error bars represent standard deviation.

Tyrosine alone with no cells does not polymerize on its own, since there was no increase over time of OD450. Interestingly, there is a statistically significant difference in the OD450s when comparing the DH5alpha cells without and with MelA transformed in them. The data indicates that, in the presence of constitutive MelA production and 2mM L-tyrosine, more melanin in produced than wild-type /E.coli. This would indicate that 1) WT E. coli can produce melanin using L-tyrosine, and 2) MelA can use L-tyrosine to produce melanin, ever moreso than WT DH5Alpha.

With this data, we intended to perform nanoparticle synthesis with L-tyrosine and MelA-transformed E. coli cells, and have these cells attach the gold nanoparticles to the surface of the E. coli cells using our FhuA-GBP fusion protein (BBa_K2045000), following the protocol created by Tsai et al [1].

[1] Tsai, Y.-J., Ouyang, C.-Y., Ma, S.-Y., Tsai, D.-Y., Tseng, H.-W., & Yeh, Y.-C. (2014). Biosynthesis and display of diverse metal nanoparticles by recombinant Escherichia coli. RSC Adv., 4, 58717-58719. Royal Society of Chemistry. Retrieved from http://xlink.rsc.org/?DOI=C4RA12805B

Sequence and Features