20.109(F09):Alkhairy+Perez

To Do List

GO HERE:[1]

Project Overview

Genetically engineering microorganism-based biosensor to detect heavy metals in water.

• Our goal is to develop a simple, inexpensive field test that can warn people or environmental authorities if dangerous levels of toxic metals 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. Currently have E.Coli strain for detecting zinc anc cadmium, or lead or Cr ^+ or nickel or mercury.
• Site some of the papers you listed

-Other technologies for detecting heavy metals (accurate but costly) (by decreasing cost) http://www.who.int/water_sanitation_health/dwq/GDW8rev1and2.pdf: look at p16-18

• Volumetric method, colorimetric method
• Electrode method
• Ion chromatography
• High-performance liquid chromatography (HPLC)
• Flame atomic absorption spectrometry (FAAS)
• Electrothermal atomic absorption spectrometry (EAAS)
• Inductively coupled plasma (ICP)/atomic emission spectrometry (AES)
• ICP/mass spectrometry (MS)

-Field Test detection methods -inaccuarate but cheap

• HPLC
• Gas chromatography (GC)
• GC/MS
• Headspace GC/MS
• Purge-and-trap GC
• Purge-and-trap GC/MS

-Benefits of this method

-Water Standards: http://www.who.int/water_sanitation_health/dwq/guidelines/en/index.html

Research Statement and Goals

Develop an E. coli system with specific photometric metal detection systems. The system response to concentrations of metal in water would be quantifiable fluorescence at different wavelengths for different metals. The system is intended to combine sensitivity with specificity and also considers bioavailability.

Specific Aims

Specific Aims:

1- Find robust E.Coli with low internal pH to with stand conditions for onsite analysis, and have high binding of metal to promoter for high levels of txn.

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

3- Fuse each promoter with (G/B/Y)Fluorescent Protein reporter gene, put fusion gene into plasmids then transform into E.Coli. Call this system 'original'. Each metal binding to its respective promoter would give peak fluorescence at a particular lambda. Call the respective wavelengths lamX, lamY, lamZ.

4- To enhance specificity and sensitivity of the system for each metal, we do the following for each promoter:

• Create library of promoter mutations at metal binding site.
• 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 its particular fluorescent protein reporter gene.

5- Insert one candidate mutant for each promoter, in different plasmids, into an in vivo system and 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 to number of cells ratio) vs. external concentration of mX or mY or mZ for each of lamX, lamY, lamZ.

6- Next, we reproduce graph above with a mixture of metals as opposed to a single metal. Ideally, graph 5 ~= graph 6, for each lambda and therefore our system is very specific.

Backup Plan: (Fill in)

Resources Needed

Deleted Info: -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