Part:BBa_K577881

pC+AraC+pBad+GFPc

This reporter part is a combination of a constitutive promoter, AraC gene, a pBad promoter (inducible with arabinose), and a GFP gene. The whole part is flanked by the regular E, X and S, P restriction sites, so it can be approached with the usual restriction digest method. The purpose of this part is to provide the user with a simple, but complete, inducible genetic device. Adding arabinose with any concentration higher than 0.01 mM seems to cause a significant expression of the GFP gene. This is useful if the user plans to build a larger plasmid construct in a short time or characterize a specific part. This part can be inserted in a cell line that does not contain any AraC in its genome, which is important if the user is not using Top 10 cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1247
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1187
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1923
  Illegal SapI site found at 961
  

XHD-WS-Wuhan-B 2019's Characterization

Aim of experiment

K577881 part is an inducible araC-pBAD promoter with GFP. This section aims to investiagte the effects of different concentrations of arabniose on GFP expression in this part and provide more detailed data for this part.

Methods

1. Growth curve of E.coli containing part K577881 in different concentrations of L-arabinose

Part K577881 was taken from iGEM distribution kit (plate 1,14F). Add 10μl ddH2O into the corresponding hole and mix well, taking 1μl to transform into E.coli DH5α. A single colony was selected from the plate and inoculated in LB broth containing chloramphenicol, cultured overnight. The overnight culture was added into the fresh LB medium containing chloramphenicol 34μg/ml at a ratio of 1:100, mixed well and divide into tubes. Different concentrations of L-arabinose solutions were added into the test tubes, respectively, so that the final concentration were 0, 500μM, 1000Μm, 2000μM, 5000μM, 1000μM and50000μM,respectively. Samples were taken every 1 hour from 0h to 8h and OD value were measured by Multiskan Spectrum Microplate Reader at 600nm wavelength.

2. GFP expression in E.coli under the control of pBAD promoter induced by different concentrations of L-arabinose

Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of L-arabinose were added into the test tube, respectively, so that the final concentration of arsenic 0, 10μM, 50μM, 100μM, 200μM, 500μM, 1000Μm, 2000μM, 5000μM, 1000μM and 50000μM,respectively. Samples were taken at overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.

3. GFP expression of E.coli under the control of pBAD promoter induced by L-arabinose at different time points

Overnight cultured bacterial solution was inoculated in LB broth containing chloramphenicol at 1:50 to expand the culture, and the experiment was started when OD600 reached 0.4-0.6. Different concentrations of L-arabinose were added into the test tube, respectively, so that the final concentration of arsenic 0, 10μM, 50μM, 100μM, 200μM, 500μM, 1000Μm, 2000μM, 5000μM, 1000μM and 50000μM,respectively. Samples were taken at different time points of 0h, 1h, 2h, 4h and overnight (16h). GFP fluorescence intensity (485 nm excitation/ 528 nm emission) and OD600 value were measured at the same time.