Protocol Mer Operon

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Protocol - Test 1: Analyze of the bacterial growth on mercury by absorbance quantification

Samples (2):

 DH5a with MerRTPA (weak regulated promoter, with repressor,

without MerB)

 DH5a with pSB1C3 (negative control)

Hg concentrations:

1) 0µg/ml – analyzed one time for each sample

2) 5µg/ml – analyzed in triplicate for each sample

1) Pre-inoculum of the colonies to be analyzed (DH5α with plasmid pSB1C3

and DH5α with genes MerRTPA in plasmid pSB1C3) in 5ml of LB broth,

both with chloramphenicol 34µg/ml.

2) Incubate at 37ºC overnight

3) Inoculate 200µl of each sample at their correspondent erlenmeyers

containing 20ml of LM broth and also chloramphenicol 34µg/ml.

4) Incubate at 37ºC

5) After 1h30min, aliquot 1ml of each sample on 1,5ml microtube

6) Add to the erlenmeyers the amount of mercury chloride of the stock to

reach 5µg/ml of concentration on both samples, since mercury

concentration at 5µg/ml will be analyzed in triplicate

7) Incubate the erlenmeyers on the shaker at 37ºC

8) Analyze absorbance of the samples at 600nm each 1h30min.

Test 2 – Mercury (Hg) tests with RTPBA and RTPA constructions

Test 1. Plaque test (Antibiogram)

Samples (3):

 DH5a with MerRTPBA (weak regulated promoter, with repressor)

 DH5a with MerRTPA (weak regulated promoter, with repressor, without

MerB)

 DH5a with pSB1C3 (negative control)

Hg concentrations:

1) 0M

2) 0,1M = 20mg/ml

3) 0,01M = 2mg/ml

4) 0,001M = 200µg/ml

5) 0,0001M = 20µg/ml

- Inoculum of the colonies to be analyzed in 5 ml of LB medium broth

containing chloramphenicol (34µg/ml)

- Overnight growth at 37ºC

- The next day, colonies dilution of 1:10 and plating 3 ml in culture medium

LB solid

- After five minutes, remove the medium excess and leave the plates to

dry for ten minutes

- Add to the plate the paper filter discs and, then, add 10 µl of the desired

HgCl2 solution

- Make control plates, adding 10µl of MilliQ water on the paper filter discs

- Incubate the plates at 37ºC for 16 hours

- Measure the diameter of the halos