Engineering | NYCU-Taipei

The First Cycle

Design

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.

Build

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

Test

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

Learn

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.

          
              The dusB promoter expression is completely different from our expectations. It shows little expression
              during the early log phase but has a sharp increase when entering the late log phase. Since the dusB
              promoter function is quite different, we could not contribute it to any possible reason. In the end, we
              decided to remove the dusB promoter from our project.
            

              The hchA promoter expressed before entering the stationary phase. The result is predictable for that the
              hchA mRNA expression shows the uprise from late log phase and reaches its peak at stationary phase. The
              point we are missing is the late log expression might be too strong so that it suppress our late log phase
              indicator.
            

              The glpABC promoter expressed more like the late log phase promoter. The result is also predictable
              since the glpABC mRNA expression reaches its peak in the middle of the mid log phase. The late log phase
              expression might be due to the time required for protein synthesize or the time required for fluorescent
              protein maturation.
            

        

Fig. 10) mRNA expression of glpA, glpB, glpC under the glpABC promoter

Fig. 11) mRNA expression of hchA under the hchA promoter

The Second Cycle

Design

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

Build

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

Test

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

Learn

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.

The Third Cycle

Design

Two hypotheses were proposed for the cause of lasting fluorescence:

          Continuous production of the fluorescent protein.
Slow degradation of fluorescent protein by E. coli DH5α

To determine which hypothesis is correct, we append the fast degradation mCherry ( BBa_K4461013) to glpABC promoter for testing.

Build

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

Test

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

Learn

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.