T7 promoter+csgA-linker-mfp5-linker-sfGFP, sfGFP as marker
Sequence and Features<h3></span>
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 47 Illegal PstI site found at 400
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 400
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 585
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INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 47 Illegal PstI site found at 400
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 47 Illegal PstI site found at 400
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 780
Introduction
This composite part is meant to express csgA-linker-mfp5-linker-sfGFP fusion genes under the T7 promoter. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing cohesive mechanical strength.
Mfp5 is mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion. Compared to T7 promoter+csga-linker-mfp5-linker-His(BBa_K3089021), we have added sfGFP to characterise the expression of the recombinant protein. It is a robustly folded version of GFP, called ‘super folder’ GFP, that folds well even when fused to poorly folded polypeptides (Waldo et al., 2006).
Characterization
The circuit of the protein BBa_K30889034
Fluorescence analysis
T7 promoter+csgA-linker-mfp5-linker-sfGFP was cloned into pET28b and transformed into E.coli BL21 (DE3).We grew 25-ml cultures of
E. coli BL21
(DE3) bearing csgA-linker-mfp5-linker-sfGFP in LB medium containing kanamycin (50 mg/ml) overnight. We grew 1000-fold dilutions in 200-μL cultures to ~0.2/0.5/0.8 OD600 nm in a 96-well plate with cover and induced them at 37℃ with 500μM IPTG for 22 h.
OD600nm and fluorescence were measured (488-nm excitation, 530-nm emission,10-nm bandpass for GFP) with a Microplate Fluorescence Reader (THERMO Varioskan Flash). Fluorescence was normalised by dividing by the OD600 nm. We continuously monitored the OD600nm and fluorescence of these four strains and plotted the graph for their growth and induced fluorescence. We added IPTG to these strains at different times of their log phase, such as OD600nm=0.2（early）, 0.5（medium）, 0.8（late）.
Results were measured by the ratio of fluorescence to OD600nm. CsgA-Mfp5-sfGFP had a relatively much poorer expression compare with sfGFP, which was used as control (Figure 1ABC).
Figure 2. Normalised fluorescence by dividing with OD600nm of sfGFP fused constructs（A-C）、OD600m(D-F) and fluorescence(G-I). Arrows indicate timing to add IPTG. Six repeats were monitored for each group and anomalies below 0 was ignored. Figure 3. Normalized fluorescence (Fluorescence/OD600nm) measurement of CsgA-linker-Mfp5-linker-sfGFP. 500uM IPTG was added into cultures when it reached OD600nm at 0.2, 0.5, 0.8 separately after a 1000-fold dilution from overnight cultures. Arrows indicate timing to add IPTG. A.U. (arbitrary units) calculated by dividing fluorescence with OD600nm value. Six repeats were monitored for each group and anomalies below 0 was ignored. Figure 4. Fluorescence curve of CsgA-linker-Mfp5-linker-sfGFP
Interestingly,
adding inducer in early stage of log phase (OD=0.2) would delay the growth of CsgA-linker-Mfp5-linker-sfGFP (Figure 1D), letting them enter to lag phase again.
During this second-lag-phase, the fluorescence was growing continuously (Figure 2G), which means proteins was still accumulating in cells. However, when growth entered log phase again, the normalised fluorescence reduced to some extent due to the rapid increase of OD600nm value.
These phenomena are generally not observed when adding IPTG in medium (OD=0.5) and late log phase(OD=0.8)( Figure 1EF).
For what we concern is the total yield, in other words, the absolute fluorescence of the culture. Results showed adding IPTG in early log phase significantly reduced overall expression, and the fluorescence reached its peak in 5 hours (Figure 4).
We concluded that we should add inducer in late log phase for higher-level expression and 5 hours’ induction was enough to get a highest yield.
Reference
Waldo, G.S. et al. (January 2006). Engineering and characterization of a superfolder green fluorescent protein. Retrieved from http://www.nature.com/naturebiotechnology
