Question

Please help

1) Make a prediction about a gene that will be present in ETEC, but not in other strains of E. coli (neither other pathogenic strains nor nonpathogenic strains). This may become evident based on what you read as you research your strain, or you may need to do some educated guess work. You also may end up developing and testing more than one hypothesis.

1. What is your hypothesis?

2) Propose actual lab experiments you would do to demonstrate the utility of the PCR assay.

1. What gene will you amplify by PCR? How will you set up the PCR?
   1. What reagents will you add?
   2. What strains will you use?
   3. What controls will you include?
   4. What cycling conditions will you use
2. Diagram the results you would obtain after gel electrophoresis.
   1. What size will the PCR product be?
   2. What lanes should have a PCR product?
   3. What conclusions can be drawn?

Answer 1

1. We will look for Heat-stable enterotoxins (STs) genes in the produced by ETEC bacteria. The size of this protein is ~7KDa (70-75 amino acids = 210- 225 bp DNA)

2. The detection of pathogenic bacteria can be done using the PCR using specific primer to STs gene.

2. The STs gene can be amplified. PCR will be set in PCR machine different reagent required in the PCR.

Reageant- Primer (forward and reverse), dNTPs, Taq Polymerase, Mg++ ions in a buffer system.

The strain (cell lysate) to be tested will be used in PCR as template.

A known pathogenic ETEC cell lysate will be used as positive control and in negative control non pathogenic bacterial will be used.

The cycling conditions will be initial denaturation at 94C for 2 min, then the 30 cycles of denaturation of 94C for 30 sec, annealing at 55C for 30sec and extenstion at 72C for 30 sec.

3.

Size will be around 210 bp

Positive control and positive test lane should have band

The conclusion is that only positive (ETEC bacteria) will show the desired band.