20.109(F09):Alkhairy+Perez

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Project Overview

Genetically engineering microorganism-based biosensor

• Our goal is to develop a simple field test that can warn people or environmental authorities if dangerous levels of toxic metals or metalloids (metal-like substances such as arsenic) are present in the environment, to which they might be exposed. The test could provide vital in helping to tackle one of the world's greatest disasters – the poisoning of tens of millions of people in Bangladesh and West Bengal, India, through naturally–occurring arsenic in their household well water. rather than through long and expensive laboratory testing

Why is this important? : http://www.uq.edu.au/news/?article=12818, http://www.associatedcontent.com/article/366604/researches_develop_new_test_for_contaminated.html

Background Information

• Past heavy metal biosensors

-Heavy Metal BioSensor-BIOMET: http://wwwa.vito.be/english/environment/environmentaltech2d.htm

• (AE1433) for zinc and cadmium;
• (AE1239) for copper;
• (AE2450) for lead;
• (AE2440) for Cr6+;
• (AE2515) for nickel;
• (CM2624) for mercury.

-Other technologies for detecting heavy metals -Benefits of this method -Journal Devoted to created biosensors: http://www.elsevier.com/wps/find/journaldescription.cws_home/405913/description#description -MicroArray for Heavy Metal Ions: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TFC-4X2DCXH-3&_user=501045&_coverDate=12%2F15%2F2009&_alid=1099894232&_rdoc=1&_fmt=high&_orig=search&_cdi=5223&_st=13&_docanchor=&_ct=288&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=a209a6a8e7caf90ef3f7009bfee4705b

Research Statement and Goals

• Develop an E. Coli system with specific photometric metal detection systems.

Specific Aims

1- Find robust E.Coli to commercialize.

2- Find promoters pX, pY, pZ regulated direct binding of metals mX, mY, mZ.

3- Fuse each promoter with (G/B/Y)FP, put into plasmids into E.Coli. Call this system 'original'. Each metal would give peak fluorescence at a particular lambda. Call the respective wavelengths lamX, lamY, lamZ.

4- For each promoter do the following

• Create library of mutations.
• SPR: immobilize mutant promoters and select for tighter binding and specificity of respective metal to find 4 in vitro candidate mutants
• Create vector with candidate mutant promoter fused to a FP reporter gene.

5-Plug the top candidate mutant, for each promoter in a different plasmid, into in vivo system, compare with 'original'. Go through all combinations of promoters mutant candidates (4*4*4) and pick best mutant combination. To compare, we specifically produce graphs w/ (intensity/number of cells) vs. [X or Y or Z]out for each of lamX, lamY, lamZ.

6- Next, we reproduce graph above with [X + Y + Z]. Ideally, graph 5 = graph 6, for each lambda and therefore our system is very specific.

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