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Part:BBa_K079050:Experience
Applications of BBa_K079050
LexA operator was tested using UV induction. Tests with difference distances from the UV lamp (3 and 4 cm) with different exposition times (1, 10 and 15 s) were done. Minimum distance and maximum time were fixed to avoid lethal UV dose and mutagenesis. After induction by UV the samples were kept for 2 hours in dark by silver paper to increase the RecA and LexA response. OD was not altered by UV irradiation but we didn't observe significant GFP levels. Probably we were not able to find the right time/distance setting. Moreover we are not sure that an uniform irradiation takes place.
Since we have not success with UV induction, we tested the correct functionality of K079050 by using hydrogen peroxide induction. It is well known [Assad et al. 2004] that hydrogen peroxide is an important reactive oxygen species (ROS). The reaction of hydrogen peroxide with transition metals imposes on cells an oxidative stress that can result in damage to cell components such as proteins, lipids and principally DNA. In E.coli low concentration of H2O2 (1-3mM) results in SOS gene induction [Imlay and Linn, 1987; Goerlich et alt. 1989]
The plasmid contained the LexA Operator was transformed into DH5alfa bacteria according to the standard protocol and one colony was picked up from the plate and let it grown O/N in 15ml tube with 5ml LB medium and ampicillin. Cultured colony was diluted in 5ml LB with antibiotic and 1ul H2O2 (11M) to have medium with 2.2mM concentration of H202 and 0.1 starting OD. As control, to prove that H2O2 has effect on the K079050, one sample of the same culture was diluted in 5ml LB medium with antibiotic without H2O2.
Northwestern Team 2019 created an analogous LexA1 part K3269004 and compared with this part, K079050 and found that LexA2 can be damaged with UV and have the reporter expressed in a plate reader. As illustrated in the graph below, GFP Fluorescence correlates with the UV-dose given to the cell, illustrating that this part works with UV induction.
LexA2 operator was tested using UV induction. Tests with difference distances from the UVC lamp (100 cm) with different exposition times (2,4, 6, 10 s) were done. We exposed the colonies in a 96-well plate and we immediately measured the fluorescence and OD in a plate reader with the orbital shaking setting on for 3 hours.
File:Lexa2graph1.png K079050 compared to K3269004 |
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