For identifying the E. coli growth status, we combined a phase-dependent promoter with a fluorescent protein to specifically express distinct color in each phase. In our design, early log phase promoter, mid-log phase promoter, late log phase promoter, and stationary phase promoter are combined with AmilCP, mCherry, yPet, mCerulean respectively, so the early log phase promoter will express color blue, mid-log phase will express color red, late log phase will express color yellow, and stationary phase will express color green. Note that the chromoprotein, AmilCP, which has slightly longer maturation time is used since the dusB expression will begin at lag phase. Here is our design.
To measure the characteristics of the potential phase specific promoters, we combined the promoters with RBS(B0034), reporter gene(YPet, mCerulean, mCherry, AmilCP) and double terminator(B0015). Since the AmilCP expression is rather hard to measure, the early log phase promoter is combined with the mCerulean for measurement.
The early log phase promoter, dusB promoter and cyoA promoter, the mid-log phase promoter, nirBDC promoter and glpABC promoter, the late log phase promoter, ansB promoter, and the stationary phase promoter, hchA promoter, rmf promoter, and slp promoter have been cloned by PCR from E. coli MG1655 genomic DNA. For more information regarding how we construct the plasmid, please visit the Result page.
The glpABC promoter, dusB promoter, and hchA promoter are the first three constructs to be successfully built. The other construct will be discussed in the next cycle for the reason mentioned in the learn part. The validation is performed by restriction enzyme digestion method with BamHI and XhoI. For more information, please visit our project Result page.
Fig. 1) The expected result for enzyme digestion confirmation for dusB-mCerulean
Fig. 2) The result for enzyme digestion confirmation of dusB-mCerulean
Fig. 3) The expected result for enzyme digestion confirmation for glpABC-mCherry
Fig. 4) The result for enzyme digestion confirmation of glpABC-mCherry
Fig. 5) The expected result for enzyme digestion confirmation for hchA-mCerulean
Fig. 6) The result for enzyme digestion confirmation of hchA-mCerulean
To evaluate if the promoter can specifically express in the desired phase, the fluorescent intensity is tested, as well as the OD value.
Fig. 7) The fluorescent intensity - time graph and OD600 value - time graph for dusB-mCerulean
Fig. 8) The fluorescent intensity - time graph and OD600 value - time graph for glpABC-mCherry
Fig. 9) The fluorescent intensity - time graph and OD600 value - time graph for hchA-mCerulean
First of all, we found that despite the promoter express phase-specific rise of gene expression, the fluorescent protein won't degrade on time. Based on the observation, the data suggests two possibilities. One is that the promoter we use isn't phased specifically but expresses from specific phases. The other is that the E. coli DH5α has a poor ability to degrade mCerulean and mCherry.
Secondly, we found that fluorescent protein is difficult to be observed by naked eye in the early log phase. It might be due to the relatively low amount of bacteria in those phases or the weak expression of the fluorescent protein.
Thirdly, the promoter's function did not work as we expected.
Fig. 10) mRNA expression of glpA, glpB, glpC under the glpABC promoter
Fig. 11) mRNA expression of hchA under the hchA promoter
Since the early exponential phase is too difficult to be observed, and the dusB promoter fails to express the desired phase, we remove the early exponential phase indicator from our project. Besides, we combine the hchA promoter with the AmilCP to delay the late log phase expression, serving as the timer. Lastly, the color of mid-log phase indicator and late log phase indicator are switched since the glpABC promoter with mCherry expresses more like the late log phase promoter, and the other mid log phase, nirBDC-mCerulean, are constructed.
Fig. 12) The construct for nirBDC-mCerulean
Fig. 13) The construct for hchA-AmilCP
The mid-log phase indicator, nirBDC-mCerulean and the stationary phase indicator, hchA-AmilCP are successfully built. The validation is also performed by restriction enzyme digestion method with BamHI and XhoI. For more information, please visit our project Result page.
Fig. 14) The expected result for enzyme digestion confirmation for nirBDC-mCerulean
Fig. 15) The result for enzyme digestion confirmation of nirBDC-mCerulean
Fig. 16) The expected result for enzyme digestion confirmation for hchA-AmilCP
Fig. 17) The result for enzyme digestion confirmation of hchA-AmilCP
To evaluate if the promoter can specifically express in the desired phase, the fluorescent intensity is tested, and as well as the OD value. For hchA-AmilCP, the photos are taken since it could not emit the fluorescence.
Fig. 18) The fluorescent intensity and OD600 - time graph for nirBDC-mCerulean
Fig. 19) OD600 value - time graph for hchA-AmilCP
Fig. 20) The photo of 9hr to 10hr for Fig. 19
The expression of the nirBDC promoter with mCerulean and the hchA promoter with AmilCP have accorded to our expectation. Therefore, the remaining question is the lasting fluorescent intensity.
Two hypotheses were proposed for the cause of lasting fluorescence:
To determine which hypothesis is correct, we append the fast degradation mCherry ( BBa_K4461013) to glpABC promoter for testing.
We successfully built our construct, and the validation is performed by BamHI and HindIII. For more information, please visit our project Results page
Fig. 21) The result for enzyme digestion confirmation of glpABC-mCherry with tag
Fig. 22) The expected result for enzyme digestion confirmation of glpABC-mCherry with tag
Both fluorescent intensity and OD of glpABC-mCherry and glpABC-mCherry-tag are tested, and the data are compared to determine which hypothesis is correct
Since we could not see the reduction of fluorescent intensity after the late-log phase, our promoter is more likely to continuously express from a specific phase. For future improvement, the protease-degradation tag system is designed to enable the fast elimination of fluorescent protein in the next phase.