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

Here, we used K577881 part, an inducible araC-pBAD promoter with GFP , to investiagte effect on different concentrations of arabniose on GFP expression in this part, and to provide a more detailed data for this part. 1. Growth curve of E.coli containing part K577881 in different concentration of L-arabinose As shown in Fig.1, the growth of bacteria was not influenced at the range of 500 -10000μM of L-arabinose. When the concentration of L-arabinose reached 50000mΜ, a little inhibition was observed, indicating a little toxicity to cells at this level. Fig.1

Fig.1 Growth curve of E. coli containing K577881 in different concentration 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 at different time points of 0h, 1h, 2h…8h at an hour interval and OD600 value were measured with a Multiskan Spectrum Microplate Reader.

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

The expression of GFP under the control of pBAD promoter at different concentrations of L-arabinose was shown in Fig.2. It is not enough to induce the expression of GFP in the range of 0-500μM. When the concentration increased form 1000μM to 5000μM, GFP was expressed in a concentration-dependt manner. The fluoresence intensity of GFP reached the peak at 5000μM. However, as the L-arabinose increased to10000μM, the expression of GFP began to decrease and at the concentration of 50000Μm, the expression was dramatically inhibited.

Fig.2

https://static.igem.org/mediawiki/parts/3/3c/T--XHD-WS-Wuhan-B--GFP1-Fig2.tiff

Fig.2 Fluorescence intensity (FL) changes of GFP protein induced by different concentration 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 As shown in Fig.3, K577881 part, the inducible araC-pBAD promoter with GFP, reacted rapidly in response to L-arabinose. The corresponding GFP fluorescence signal can be detected at 1000μM within an hour. As time increased, the expression of GFP increased. Fig.3

Fig3. Fluorescence intensity (FL) changes of GFP protein induced by different concentration of 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.

summary

K577881 part, the inducible araC-pBAD promoter with GFP, is sensitive and fast. The GFP began to express at 1000μM within an hour. The optimal concentration range for arabinose-induced araBAD promoters is 1000-5000 μM, which is dose-dependent and increases with time.