Team:Concordia/Safety/SOP/Recombinant
Nanoparticle Attachment: Recombinant
Materials:
-Plate of DH5α E coli strain expressing Rhizobium etli CFN42 tyrosinase (MelA) plasmid
-Double distilled water (ddH2O)
-Micropipets and tips
-1-2 culture tubes
-LB media
-IPTG solution
-L-DOPA solution
-1.5mL microfuge tubes
-Au3+ (gold) salt → prepare into 2mM solution
**Perform all steps in BIOSAFETY CABINET, except for boiling which is done under the FUME HOOD
Protocol:
1. From a plate of MelA-transformed DH5α E.coli, aseptically pick 1-2 colonies and inoculate in 5mL liquid medium (LB) in culture tube(s). Place the tube(s) on a shaker at 250 speed and 37oC temperature overnight.
Production of EuMel
2. Remove the culture tubes from the shaker and work on a bench at room temperature.
3. Induce the exponential phase of the DH5α E.coli and allow production of EuMel by:
inducing with 100 mM of IPTG and 1 mM L-DOPA for 12 hours. Then, cells were centrifuged at 12,225 g for 15 min and the supernatant was retained for the following reaction
4. Incubate the supernatant with 2.0 mM of Au3+ (HAuCl4．3H2O,)
Collection of EuMel produced
5. Use a micropipet to transfer aliquots of the cell solution to 1.5mL microfuge tubes.
the supernatant was collected (centrifuged cells at 12,225 g, 15 min). Before measuring optical density at 400 nm, the solutions were adjusted to pH=7. The concentration of melanin was calculated from standard curves of chemically produced melanin (Sigma) as references (Fig. S2)
Synthesis of nanoparticles
6. Recombinant E. coli were inoculated from single colonies and freshly diluted (2%) in M9 medium supplemented with 0.2% glucose and kanamycin (50 g/mL)
induced with 100 nM of IPTG, 1 mM L-DOPA and 0.2% arabinose, followed by adding 2mM Au3+ (HAuCl4．3H2O) and further cultivation for 2 - 3 h
7. Cells were harvested by centrifugation at 12,225 g for 1 min and washed three times with deionized water
8. An aliquot of the nanoparticle solution can be subjected to UV-VIS spectrophotometric analysis to detect presence of nanoparticles.
-Expected absorbance at 535nm
Source: Tsai, Y-J., Ouyang, C-Y., Ma, S-Y., Tsai D-Y., Tseng, H-W., and Yeh, Y-C. (2014) Biosynthesis and display of diverse metal nanoparticles by recombinant Escherichia coli. RSC Adv., 4:58717-58719. Retrieved from http://pubs.rsc.org/en/Content/ArticleLanding/2014/RA/c4ra12805b#!divAbstract
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