Construct Plasmid for Growth Phase Indicator
We applied a series of existing plasmid constructs which were kindly provided to us by our supervisor for the construction of our growth phase indicator product. Each plasmid consists of a constitutive promoter (BBa_J23100), a color indicator gene, and double terminators (BBa_B0015). First of all, pSB1C3 plasmids containing our selected reporter gene (mCerulean, mCherry, yPet, AmilCP) were confirmed by restriction enzyme digestion (Fig. 1). In the following steps, these plasmids will be used to construct our newly assembled plasmid, which replaces the constitutive promoter with our phase-dependent promoter.
Fig. 1) The result of enzyme digestion confirmation of YPet, mCerulean, mCherry, AmilCP
Fig. 2) The expected result of enzyme digestion confirmation
Fig. 3) The construct of YPet, mCerulean, mCherry, AmilCP
Then, we extracted the genomic DNA from E. coli K-12 MG1655
Fig. 4) the result of E. coli K12 Mg1655 gDNA extraction
The potential phase-dependent promoters, dusB, nirBDC, glpABC, ansB, rmf and hchA promoter, were cloned from gDNA of E. coli K-12 MG1655. Note that the length of the sequence that is cloned is larger than the promoter we used. This is to prevent the amplification of unintended bands. After the cloning from gDNA of E. coli K-12 MG1655, overlapping sequences were added by PCR on both sides of the promoter sequence, which allows the performance of Gibson assembly.
Fig. 5) The result of PCR cloning of each promoter from gDNA
Fig. 6) The result of PCR cloning to append overlapping site to promoter
pSB1C3 plasmids containing the fluorescent-terminator sequence were cloned into two pieces by PCR and are added with overlapping sequences. This will prevent the situation that the construct size is too similar to the original plasmid and causes low yield after Gibson transformation
Fig. 7) The construct for pSB1C3 plasmids containing the constitutive promoter (J23100), RBS (BBa_B0034), and fluorescent-terminator sequence
Fig. 8) The sequence for vector 2, which contains RBS, reporter gene and the double terminator
Fig. 9) The result of PCR cloning for each vector 2, containing a different reporter gene. The expected result for YPet is 1072 bp, for mCerulean is 1078 bp, for AmilCP is 1027 bp, for mCherry is 1040 bp
Fig. 10) The sequence for vector 1, which is the pSB1C3 backbone
Fig. 11) The result of PCR cloning for vector 1. The expected result is 1874 bp
Early Log Phase
The potential early log phase promoter, dusB promoter, was combined with RBS-mCerulean-double terminator sequence and pSB1C3 backbone sequence by Gibson assembly.
Fig. 12) The construct for dusB-mCerulean
The plasmid was then transformed into E. coli DH5α competent cells, and the successful colonies were checked by enzyme digestion.
Fig. 13) The expected result for enzyme digestion confirmation for dusB-mCerulean
Fig. 14) The result for enzyme digestion confirmation of dusB-mCerulean
Mid Log Phase
The potential mid log phase promoter, nirBDC promoter, was combined with RBS-mCerulean-double terminator sequence and pSB1C3 backbone sequence by Gibson assembly
Fig. 15) The construct for nirBDC-mCerulean
The plasmid was then transformed into E. coli DH5α competent cells, and the successful colonies were checked by enzyme digestion.
Fig.16) The expected result for enzyme digestion confirmation for nirBDC-mCerulean
Fig. 17) The result for enzyme digestion confirmation of nirBDC-mCerulean
The potential mid log phase promoter, glpABC promoter, was combined with RBS-mCherry-double terminator sequence and pSB1C3 backbone sequence by Gibson assembly. Note that it turns out that glpABC promoter expression more likely occurs during the late log phase.
Fig. 18) The construct for glpABC-mCherry
Fig.19) The expected result for enzyme digestion confirmation for glpABC-mCherry
Fig. 20) The result for enzyme digestion confirmation of glpABC-mCherry
Late Log Phase
The potential late log phase promoter, ansB promoter, was only cloned from the gDNA since the glpABC expression represents the pattern of a late log phase promoter.
Stationary Phase
The potential stationary phase promoter, hchA promoter, was combined with either RBS-mCerulean-double terminator sequence or RBS-AmilCP-double terminator sequence and pSB1C3 backbone sequence by Gibson assembly. The two different colors were both constructed to give a more observable option.
Fig. 21) The construct for hchA-mCerulean and hchA-AmilC
The plasmid was then transformed into E. coli DH5α competent cells, and the successful colonies were checked by enzyme digestion.
Fig. 22) The expected result for enzyme digestion confirmation for hchA-AmilCP
Fig. 23) The result for enzyme digestion confirmation of hchA-AmilCP
Fig. 24) The expected result for enzyme digestion confirmation for hchA-mCerulean
Fig. 25) The result for enzyme digestion confirmation of hchA-mCerulean
Functional Test for Promoter and the Validation
The fluorescence intensity and OD value were both tested to determine whether our promoter expressed in each specific phase.
dusB
Although literature evidence strongly supports the phase-dependent expression of the dusB-fis promoter during early-exponential phase, the measured fluorescent intensity data demonstrates a pattern that closely resembles a constitutive promoter.
Fig. 26) The fluorescent intensity - time graph and OD600 value - time graph for dusB-mCerulean
nirBDC
The expression curve of the nirBDC promoter occurs during the mid-exponential phase, which was the same as we expected.
glpABC
The glpABC promoter did not express from the mid-exponential phase as we expected. However, its expression perfectly matches the late-exponential phase. Therefore, the glpABC promoter was used to indicate the late-exponential phase.
Fig. 27) The fluorescent intensity and OD600 - time graph for nirBDC-mCerulean and glpABC-mCherry
hchA
The hchA promoter is continuously expressed, and shows the rather fast incline from the late-exponential phase. We combined it with AmilCP, which has a slightly longer maturation time (1h), to delay its expression and enable the hchA promoter to indicate stationary phase. Although this is not the optimal method for indicating stationary phase, our project is still workable with this part. Despite the fact that we could not find the stationary phase-dependent promoter ourselves, the MIT 2006 iGEM has developed a stationary phase promoter osmY which has been recorded on the iGEM Parts of Registry.
Fig. 28) The fluorescent intensity - time graph and OD600 value - time graph for hchA-mCerulean
Fig. 29) OD600 value - time graph for hchA-AmilCP
Fig. 30) The photo of 9hr to 10hr for Fig. 29
Since the fluorescence will remain after the desired phase, ssrA degradation tags were attached to the fluorescent protein to increase its degradation rate in E. coli. This allowed us to test whether the lasting fluorescence is caused by the continuous expression of the fluorescent protein or the slow degradation rate of fluorescent protein by E. coli. The possible results are shown in (Fig. 31).
Fig. 31) The possible result when the tag is attached to fluorescent protein
In order to attach the degradation tag behind the fluorescent protein, point mutation is performed to prevent the effect of the stop codon. Confirmation of whether the point mutation has been successfully performed is done by enzyme digestion since the cutting site, HindIII, is deleted after point mutation.
Fig. 32) The result for enzyme digestion confirmation of glpABC-mCherry mutation
PCR is performed to append cutting sites, HindIII and EcoRI, between fluorescent protein and terminator. The tag with HindIII and EcoRI cutting sites is synthesized due to its short length. The ligation is then performed. The confirmation is performed by restriction enzyme digestion method with BamHI and EcoRI.
Fig. 33) The result for enzyme digestion confirmation of glpABC-mCherry with tag
The result suggests that our promoter is specific express from desired phases. Therefore, the degradation system is designed to prevent the lasting fluorescent intensity. However, due to the time limit, we are unable to finish it.
Fig. 34) The result for fluorescent intensity and OD measurement of glpABC-mCherry with tag
Ligation of Each Phase to Form the Three-Phases Indicator
The mid-exponential phase, late-exponential phase, and stationary phase indicator sequence are cut with NotI/XhoI, NotI/PstI and PstI/XhoI respectively. The ligation products are confirmed by PCR with primers located at nirBDC promoter and glpABC promoter.
Fig. 35) The result for PCR confirmation of nirBDC-mCerulean, glpABC-mCherry, and hchA-AmilCP ligation
fluorescence intensity are tested and pictures are taken as well
Fig. 36) The result for fluorescent intensity and OD measurement of ligation
Fig. 37) The photo of 10 hr to 11 hr from Fig. 36 to show AmilCP expression
Validation of Our Growth Phase Indicator
For validation, we use the ribosome dynamic as the indicator for the growth phase since it shows the growth phase dependent pattern. MCrg, the generous gift from Dr. Nikolay, are used as the biomarker for ribosome dynamics by expressing fluorescent-labeled ribosomal proteins L19-GFP and S2-mCherry. We made it as the competent cell to check whether the color change of our indicator could correspond to the growth phase indicated by ribosome dynamics. However, due to the time limit, the successful colonies are not yet to be found.
Fig. 38) the MCrg competent cell are culture on LB plate to make sure it is not dead during the competent cell preparation
Measurement Data for Different Cultivate Condition
The measurements start with OD600=0.05 and total LB+CM volume of 50ml, the OD600 are recorded by a microplate reader. The sample is the E. coli DH5α with pSB1C3 plasmid serving as the CM resistance. The loading volume is 100 μl, and the cultivating environment is 200rpm and 37°C
Different Quantity of Bacteria That are Cultured
Fig. 39) The result for OD measurement with a different initial quantity of bacteria. The meaning of the data can be seen in the Modeling page
Different Status of Bacteria That are Cultured
Fig. 40) The result for OD measurement when regrowing bacteria from different growth phases. The meaning of the data can be seen in the Modeling page.
Native Color Indicator
First, the part K1789003 is confirmed using Colony PCR. It was run with primer VF2/VR and confirmed with 1 % Agar gel electrophoresis for 45 min, the result (Fig. 41) expected to be 800 bp.
Fig. 41)
The fluorescent intensity of Part K1789003 is observed using Tecan Elisa Reader and its data is collected as shown below (Fig. 42). We then narrowed down the Excitation/Emission range of Part K1789003 between 470nm/510~530nm and reobserved. Its data is collected as shown below (Fig. 43). In both data, in order to identify the background value, LB+CM is used as the source of A3 well.
Fig. 42)
Fig. 43)
We later concluded that the liquid culture of DH5α bacteria strain would be a better source for the background value of original cell culture than LB+CM so fluorescent intensity of Part K1789003 is reobserved with K1789003/DH5α/LB+CM, thus our observation data is made more complete.
After confirmation, we planned of using K608008 (J23104 + RBS) as template and RF cloning method, K1789003(sGFP1) would be inserted to replace the original GFP.
Cell Division Indicator
To indicate cell division, we clone both of the ftsZ and ftsQ promoters from E. coli DH5α gDNA since they show oscillation during the cell cycle.
Fig. 41) The result of PCR cloning of ftsQ promoter and ftsZ promoter
Plasmid Construction and Functional Test
Currently, we successfully constructed both of the potential promoters for our cell division indicator system. However, due to time limitations, we have not combined it with the reporter gene. The functional tests of these plasmids had not been performed yet.