Team:H14Z1 Hangzhou/Contribution
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Contribution
Improved functional characterization of BBa_J04450
Purpose
Previous experiments had shown that BBa_J04450-transformed E.coli BL21(DE3) would turn red color under natural light after 18 hours due to leaking expression. This part is useful for characterizing the expression of different proteins using the lacI sensitive promoter. For better application of this promoter in controlling protein expression, the effects of different hosts under various conditions were investigated systematically. The target hosts include E. coli BL21(DE3) and E. coli MC1061. The target culture conditions were temperature, inducer concentration and medium volume in the flask.
Experiment design
Exploring the effect of temperature on mRFP expression
1. Inoculate 5 ml LB with monocolony (BL21(DE3)/MC1061) from the plate. Grow the cells overnight at 37 ℃，200 rpm.
2. Dilute pre-culture 1:100 into 5ml LB (BL21(DE3)/MC1061) and cultivate at different temperature (26, 30, 34, 37 ℃), 200 rpm, respectively.
3. Measure the fluorescence and OD600 every 4 hours.
Exploring the effect of medium volume on mRFP expression
1. Inoculate 5 ml LB with monocolony (BL21/MC1061) from the plate.Grow the cells overnight at 37 ℃, 200 rpm.
2. Dilute pre-culture 1:100 into 30ml, 50ml, 70ml LB (BL21(DE3)/MC1061) and cultivate at 37 ℃, 200 rpm.
3. Measure the fluorescence and OD600 every 2 hours.
Exploring the effect of concentration of IPTG on mRFP expression
1. Inoculate 5 ml LB with monocolony(BL21(DE3)/MC1061) from the plate. Grow the cells overnight at 37 ℃, 200 rpm.
2. Dilute pre-culture 1:100 into 5ml LB (BL21(DE3)/MC1061), cultivate at 37 ℃, 200 rpm. Protein expression are induced using 0.1, 0.3, 0.5, 0.8, 1.0 mM IPTG, respectively.
3. Measure the fluorescence and OD600 every 4 hours.
Results
Exploring the effect of concentration of IPTG on mRFP expression
There are some differences of growth rate at different temperatures for both E. coli strains (BL21(DE3), MC1061) (Fig.1). However, the differences of the fluorescence intensity at different temperature is much higher than those of the growth rate at various temperatures by using two E. coli cells (Fig 2).
There are some differences of growth rate at different temperatures for both E. coli strains (BL21(DE3), MC1061) (Fig.1). However, the differences of the fluorescence intensity at different temperature is much higher than those of the growth rate at various temperatures by using two E. coli cells (Fig 2).
Fig 1 Effect of different temperature on cell growth
Fig 2 Effect of different temperature on mRFP expression
Effects of medium volume on mRFP expression
The mRFP expression of two strains showed the same trend in different volume of medium in the flask. After 6 hours, the fluorescence intensity in 30 ml medium-containing flask is significantly higher than others, indicating that mRFP express fastest in this volume scale (Fig 3). This suggest that dissolved oxygen is an important factor which will affect the protein expressin and cell growth, since the lower medium volume in the same type flask will bring about higher level of dissolved oxygen in the medium.
Fig 3 Effects of medium volume in the flask on mRFP expression
Effects of IPTG concentration on mRFP expression
Our results showed that significant fluorescence differences were not observed by using different IPTG induction (Fig 3). However, it is interesting that the fluorescence intensity of BL21(DE3) after IPTG induction are higher than those of MC1061, not similar to the trend in our previous experiment (Fig 2). It might be explained that the expression of mRFP can be accelerated by IPTG inductin in BL21(DE3) with regard to other E. coli hosts.
Fig 4 Effect of different IPTG concentration on mRFP expression
The photos of culture in the flasks under different cultivation conditions
