Characterization
Characterization of K577881
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.
Results
Fig.1 Growth curve of E. coli containing K577881 in different concentrations of L-arabinose.
As shown in Fig.1,bacterial growth was not affected when the concentration of L-arabinose ranges from 500 to 10000μM. When the concentration of L-arabinose reached 50000mΜ, a slight inhibition was observed, indicating that the toxicity to the cells was weak at this level.
Fig.2 Fluorescence intensity (FL) changes of GFP protein induced by different concentration of L-arabinose.
Fig.2 shows the expression of GFP under the control of pBAD promiter under different concentrations of L-arabinose. It is not sufficient to induce the expression of GFP in the range of 0-500μM. When the concentration increased to 5000μM, GFP was expressed in a concentration-dependent manner. The fluoresence intensity of GFP reached a 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.
Fig3. Fluorescence intensity (FL) changes of GFP protein induced by different concentration of 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.
Summary
the K577881 part, which has an inducible araC-pBAD promoter with GFP, is sensitive and rapid. GFP began to express at 1000μM within an hour. The optimal concentration range for arabinose-induced araBAD promoter is from 1000 to 5000 μM, which is dose-dependent and increases over time.