The ability to assay whether a chassis is actively transcribing its circuit to make proteins is crucial for testing the efficacy of a fieldable construct. Bacteria have two main life states: exponential growth, during which they reproduce and express their circuits with ease, and stationary phase, during which they cease most non-essential metabolic activity. Since stationary phase is induced by inopportune environments, such as metabolite shortage, most bacteria in nature exist in stationary phase (Jaishankar 2000). This is a major problem for fieldable synthetic biology, as constructs that work perfectly in the lab may stop expressing their circuits when introduced into their deployment sites. In order to assay how a circuit will behave in nature, constructs should be tested in the lab while in stationary phase. To this end, William and Mary has designed two stationary phase detection constructs, using red and green fluorescence as outputs. These composite parts are an improvement of MIT iGEM 2006’s composite part BBa_J45995, and are included in the iGEM Registry as parts BBa_K4174001 (red fluorescence) and BBa_K4174002 (green fluorescence), where you can find their full sequences. Below we have outlined information about [1] the original MIT iGEM 2006 composite part, [2] our improved parts, [3] our protocols for construct assembly, [4] our protocol for testing our constructs, and [5] results demonstrating our constructs exhibiting stronger fluorescence than the original construct.
1. Original Part: MIT iGEM 2006 osmY Stationary Phase Detection Circuit
The original composite part, designed by MIT iGEM 2006, can be found on the iGEM Registry as part BBa_J45995. This construct consists of the following basic parts: an osmY promoter, ribosome binding sequence BBa_B0030, a green fluorescence protein (GFP) coding region, and terminator BBa_B0015. Between the promoter and RBS, RBS and coding region, and coding region and terminator, the DNA sequence contains scar sequences from assembly.
The promoter selected was an osmY promoter, as this promoter is induced by the cell’s entry into stationary phase. In typical E. coli cells, osmY, which helps cells transition into stationary phase when under osmotic or metabolic stress, is not produced during exponential growth phase but is produced during stationary phase. Specifically, the osmY promoter is induced by rpoS (ribosome polymerase sigma S) at the onset of stationary phase (Chang 2002). Because the coding region in this construct is GFP, this construct fluoresces green once the cell enters stationary phase.
2. Improved osmY Stationary Phase Detection Circuits
A. osmY-sfGFP Stationary Phase Detection Construct (BBa_K4174002)
We improved this part by [a] increasing fluorescence by replacing GFP with superfolder GFP (sfGFP), [b] removing non-functional scar sequences, [c] making the sequence compatible with a new assembly method (Type IIS), and [d] adding unique nucleotide sequences.
a. Replacing GFP with sfGFP and Changing the RBS
Superfolder GFP (sfGFP) is an improved version of GFP which folds more readily and precisely in Escherichia coli, allowing for more efficient, bright, and accurate assays (Pédelacq 2006). We sourced our sfGFP sequence from Ceroni et al. (2015). This sfGFP sequence is codon-optimized for E. coli and was designed by Ceroni et al. (2015) using DNA2.0 for high levels of expression in E. coli (Ceroni et al. 2015). In order to ensure that our ribosome binding sequence (RBS) was compatible with our coding region, we also replaced the original RBS with an RBS used by Ceroni et al. (2015) with their sfGFP sequence. This RBS was designed by Ceroni et al. using the RBS Calculator (Arpino et al. 2013).
b. Removing Non-functional Scar Sequences
The original construct sequence has scar sequences present from assembly. These nonfunctional units were removed by our team as they are purely artifacts of biological assembly.
c. Making the Construct Type IIS Assembly Compatible
All constructs uploaded to the iGEM Registry that are considered for the iGEM Competition must be compatible with either Type IIS Assembly or BioBrick RFC 10 Assembly. The original MIT iGEM 2006 construct was only compatible with RFC 10, but after altering the sequence as described above to create our composite part, it is now compatible with both Type IIS Assembly and RFC 10 Assembly. Increasing the options for assembly methods compatible with our construct will help make its construction more accessible to researchers.
d. Addition of Unique Nucleotide Sequences (UNSs)
We added unique nucleotide sequences (UNSs) 1 and 10 (Torella et al., 2014) to make this part compatible with Gibson Assembly using the pSB1C3 backbone, as we also added UNS1 and UNS10 to this backbone.
B. osmY-mRFP1 Stationary Phase Detection Construct (BBa_K4174001)
We improved this part by [a] offering a new fluorescence option to researchers by replacing GFP with monomeric red fluorescence protein (mRFP1), [b] making the sequence compatible with a new assembly method (Type IIS), and [c] adding unique nucleotide sequences.
a. Replacing GFP with mRFP1
We elected to use monomeric red fluorescent protein (mRFP1) as opposed to the original GFP in order to provide an alternative reporter to GFP for the detection of stationary phase in bacterial cells. mRFP1 is a monomer, has a rapid maturity rate, and has minimal spectral overlap with GFP compared to the wild-type red fluorescent protein DsRed (Campbell et al. 2002). Creating alternative versions of fluorescent bioreporters using proteins with different excitation and emission wavelengths allows researchers to assay multiple parameters at a time, as different fluorescent assays conducted simultaneously must use proteins with different absorption spectra in order for researchers to differentiate between them.
b. Making the Construct Type IIS Assembly Compatible
All constructs uploaded to the iGEM Registry that are considered for the iGEM competition must be compatible with either Type IIS Assembly or BioBrick RFC 10 Assembly. The original MIT iGEM 2006 construct was only compatible with RFC 10 Assembly, but after altering the sequence as described above to create our composite part, it became compatible with both Type IIS Assembly and RFC 10 Assembly. Increasing the options for assembly methods compatible with our construct will help make its construction more accessible to researchers.
c. Addition of Unique Nucleotide Sequences (UNSs)
We added unique nucleotide sequences (UNSs) 1 and 10 (Torella et al., 2014) to make this part compatible with Gibson Assembly using the pSB1C3 backbone, as we also added UNS1 and UNS10 to this backbone.
3. Construct Assembly
In order to assemble the original MIT iGEM 2006 construct (BBa_J45995) along with our improved constructs, we selected to use Gibson Assembly. Our inserts (described in the design section above) were synthesized by IDT and were flanked by unique nucleotide sequences (UNSs) 1 and 10 (Torella et al. 2014). Our backbone, pSB1C3, was also flanked by UNSs 1 and 10. We isolated this backbone from a 2018 William and Mary iGEM construct using PCR primers that anneal to the UNS sequences, then performed a gel extraction and PCR purification before utilizing the backbone for Gibson Assembly. Please download our protocols for PCR, gel extraction, PCR purification, transformation of E. coli and Gibson Assembly below.
Download Protocols Below:
4. Protocol for Testing Parts
To test the effectiveness of our improved parts, our team grew all three constructs in E. coli NEB5-α cells in our plate reader. They were grown at 37°C, and were continuously shaking in a plate reader (with OD600 and fluorescence measurements taken every 10 minutes). Although the cultures were grown for around a total of 19 hours and 35 minutes, the graphs shown below only include measurements taken starting from around the 10 hour and 5 minute mark. In addition, the graphs below represent the averages of fluorescence measurements (normalized to OD600) taken from two experiments. However, the inoculation process differed between these two experiments. For one of our experiments, we diluted a culture grown overnight in 4 mLs of LB to an OD of 0.1, then loaded the culture into wells to grow overnight. For the other experiment, we inoculated into 1 mL of LB, waited roughly 30 minutes, and loaded the culture into the well plates to grow. For both experiments, 200 uLs of culture was loaded into each well. Also, for both experiments, green fluorescence was measured using an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and red fluorescence was measured using an excitation wavelength of 584 nm and an emission wavelength of 610 nm. Please download our raw fluorescence data and protocols for preparation of LB media, inoculation, and plate reader testing below.
Download Raw Fluorescence Data Below:
Download Protocols Below:
- Preparation of LB Media Protocol
- Inoculation Protocol
- Fluorescence Intensity Testing Protocol
- Plate Reader Protocol
5. Results
BBa_K417002 Characterization (osmY-sfGFP Construct)
To test the effectiveness of our osmY-sfGFP construct (BBa_K4174002), our team transformed the original MIT iGEM 2006 construct (BBa_J45995), our improved osmY-sfGFP construct (BBa_K4174002), and our improved osmY-mRFP1 construct (BBa_K4174001) in E. coli NEB5-α in a plate reader. They were grown at 37°C, and were continuously shaking in a plate reader (with OD600 and fluorescence measurements taken every 10 minutes). Although the cultures were grown for around a total of 19 hours and 35 minutes, the graph shown below only includes measurements taken starting from around the 10 hour and 5 minute mark. In addition, the graphs below represent the averages of fluorescence measurements (normalized to OD600) taken from two experiments. However, the inoculation process differed between these two experiments. For one of our experiments, we diluted a culture grown overnight in 4 mLs of LB to an OD of 0.1, then loaded the culture into wells to grow overnight. For the other experiment, we inoculated into 1 mL of LB, waited roughly 30 minutes, and loaded the culture into the well plates to grow. For both experiments, 200 uLs of culture was loaded into each well. Also, for both experiments, green fluorescence was measured using an excitation wavelength of 485 nm and an emission wavelength of 528 nm.
The data represented in the graph above only includes measurements taken starting from around the 10 hour and 5 minute mark (out of a total growth time of about 19 hours and 35 minutes). In addition, the data shown represents the averages of fluorescence measurements (normalized to OD600) taken from two experiments. Please note that the inoculation process differed between these two experiments (see the "Protocol for Testing Parts" section of this page for details).
As seen in the graph above, both the osmY-sfGFP (BBa_K4174002) and MIT iGEM 2006 osmY (BBa_J45995) constructs enter stationary phase right before 14 hours, but our improved osmY-sfGFP construct (BBa_K4174002) is much more fluorescent. The other constructs are our osmY-RFP construct (BBa_K4174001) and untransformed E. coli cells, both of which serve as negative controls for green fluorescence.
As seen in the image above, qualitative results reveal that our improved constructs are more fluorescent than the original construct (BBa_J45995). Here, bacterial cells transformed with our osmY-sfGFP (BBa_K4174002) construct are on the far right, and are visibly more green than cells transformed with the original MIT iGEM 2006 construct (BBa_J45995).
BBa_K417001 Characterization (osmY-mRFP1 Construct)
To test the effectiveness of our osmY-mRFP1 construct (BBa_K4174001), our team transformed the original MIT iGEM 2006 construct (BBa_J45995), our osmY-mRFP1 construct, and our osmY-sfGFP construct (BBa_K4174002) into E. coli NEB5-α cells and grew the various transformants in a plate reader. They were grown at 37°C, and were continuously shaking in a plate reader (with OD600 and fluorescence measurements taken every 10 minutes). Although the cultures were grown for around a total of 19 hours and 35 minutes, the graph shown below only includes measurements taken starting from around the 10 hour and 5 minute mark. In addition, the graphs below represent the averages of fluorescence measurements (normalized to OD600) taken from two experiments. However, the inoculation process differed between these two experiments. For one of our experiments, we diluted a culture grown overnight in 4 mLs of LB to an OD of 0.1, then loaded the culture into wells to grow overnight. For the other experiment, we inoculated into 1 mL of LB, waited roughly 30 minutes, and loaded the culture into the well plates to grow. For both experiments, 200 uLs of culture was loaded into each well. For both experiments, red fluorescence was measured using an excitation wavelength of 584 nm and an emission wavelength of 610 nm. The values for red fluorescence intensity normalized to OD600 values are reported below.
The data represented in the graph above only includes measurements taken starting from around the 10 hour and 5 minute mark (out of a total growth time of about 19 hours and 35 minutes). In addition, the data shown represents the averages of fluorescence measurements (normalized to OD600) taken from two experiments. Please note that the inoculation process differed between these two experiments (see the "Protocol for Testing Parts" section of this page for details).
Based on the graph above, the bacterial cells engineered with our osmY-mRFP1 (BBa_K4174001) construct appear to have entered stationary phase around 16 hours. As seen in the graph, our osmY-mRFP1 (BBa_K4174001) construct produces more red fluorescence than the original construct (BBa_J45995) (as expected since the original construct was a GFP construct). The other measurements taken are for our osmY-sfGFP construct (BBa_K4174002) and untransformed E. coli NEB5-α cells, both of which serve as negative controls for red fluorescence.
The fluorescence intensity of the untransformed cells appears higher than that of cells transformed with our osmY-mRFP1 construct due to our normalization process. Although the raw fluorescence values of the untransformed cells were consistently lower than the raw fluorescence values of the osmY-mRFP1 circuit, the OD600 values of the untransformed cells were much lower than the OD600 values of the cells transformed with osmY-mRFP1. Therefore, when we normalized by dividing raw fluorescence by OD600, the fluorescence intensity of the untransformed cells appeared to be higher than that of the osmY-mRFP1 transformants.
As seen in the image above, qualitative results reveal that our osmY-mRFP1 construct (BBa_K4174001) is visibly red, unlike our osmY-sfGFP construct (BBa_K4174002) and the original MIT iGEM 2006 construct (BBa_J45995). Here, bacterial cells transformed with our osmY-mRFP1 construct are on the far left alongside the other two cultures transformed with the osmY-sfGFP (BBa_K4174002) and MIT iGEM 2006 osmY (BBa_J45995) constructs.
The p values below are from two-sample unequal variance t-tests that use a two-tailed distribution. These tests were performed on six technical replicates from two different experiments in which the inoculation process differed. The data being compared is described in the left hand column, and the p values are provided in the right hand column.
References
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