Team:William and Mary/Engineering
Engineering Success
The W&M 2021 iGEM team had significant engineering success this season. We are able to demonstrate engineering success with circuits to assess burden at all levels of the central dogma. These include the following circuits: 3WJ Dimeric Broccoli Circuit, translational burden sensors, phosphorylation sensors, lon protease sensor and hslVU protease sensor.
We were also able to demonstrate engineering success with circuits that assess orthogonality beyond burden. These include: dnaK and dnaJ heat shock protein sensor and clpB (ATPase) sensor. As explained in detail below, our team followed the engineering cycle repeatedly throughout our project in order to design, construct, and test our circuit system.
Improvement of a Part
This year, our team was able to improve part BBa_K769001 from iGEM12_Tokyo-NoKoGen. This part consists of the ompC promoter, the B0034 RBS, GFP, and the B0015 terminator. To improve this part, our team created construct BBa_K3773515 which replaces GFP with an sfGFP sequence from Ceroni et al. 2015.
The ompC promoter is a part of the EnvZ/OmpR system. In E. coli , this system senses changes in the osmolarity of a cell's surrounding environment. In high osmolarity environments, ompC is produced. (Cai et al., 2002) Therefore, in order to confirm the functionality of our circuit, our team grew bacteria (E. coli BL21 and NEB5-alpha) transformed with this construct overnight in LB with a 20% concentration of sucrose. The next morning, after around 16 hours of growth, we used a plate reader to quantify the fluorescence output of our circuit. We found that our construct produced an average output of 73,913.25 RFU and 32,334 RFU in NEB5-alpha and BL21, respectively. To determine if our circuit was an improved version of the iGEM12_Tokyo-NoKoGen iGEM construct, our team compared our data to the data that Universitas Indonesia published on the parts page for part BBa_K769001. When grown in media with approximately the same osmolarity, their fluorescence outputs averaged 7,505.25 RFU in DH5-alpha (a similar strain to NEB5-alpha) and 7,408.72 in BL21. Therefore, our part was able to provide a greater fluorescence output than the original part, proving that our team was able to improve the original composite part.
Engineering Success of Individual Circuits
For clarity, we have divided our circuits into the following categories, although they may fit into multiple categories.
Transcriptional Burden Sensor Circuits
F30-Broccoli Circuits
Design: Using the F30-Broccoli aptamer, we designed two transcriptional burden sensors. Our first design, WM21_011, places control of F30-Broccoli expression under the strong, constitutive Anderson promoter BBa_J23119, without incorporation of any RBS or spacers. Our second design, WM21_012, places control of F30-Broccoli expression under the strong, constitutive P70a promoter without incorporation of any RBS or spacers. In both circuits, F30-Broccoli is directly followed by the strong, synthetic, bidirectional terminator BBa_B1006.
Build: In order to construct these circuits, we ordered both inserts (including promoter, F30-Broccoli aptamer sequence, and terminator) flanked by unique nucleotide sequences (UNSs) 1 and 10, as gBlocks from IDT. We then used Gibson Assembly to join our insert with the vector pSB1C3 flanked by UNSs 1 and 10. Following Gibson Assembly, we transformed our transcriptional burden sensor circuits individually into E. coli DH5-alpha using a standard heat shock method.
Test: After transformations, we attempted to confirm the functionality of our circuits. We did this by growing a culture of transformed DH5-alpha cells overnight (37°C and 250 RPM), diluting to an OD600 of 0.4, and following 2-4 hours of growth, incubating the cells with the aptamer-activating dye DFHBI-1T at room temperature for 45 minutes. After the 45-minute incubation period, we tested for aptamer fluorescence in our culture using a plate reader and comparing its fluorescence to samples of untransformed cells and LB (negative controls) and samples of our translational burden sensor circuit (positive control). However, we were unable to confirm the functionality of our circuits, as the fluorescence levels of our samples transformed with transcriptional burden sensor circuits was similar to that of the negative controls. This is most likely due to synthesis difficulties, as F30-Broccoli itself contains repeats within its sequence as well as a hairpin within its sequence. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
3WJ Dimeric Broccoli Circuit
Design: We used the 3WJ dimeric Broccoli (3WJdb) circuit (pUC19-P70a-3WJdB-T), which was designed and constructed entirely by researchers Burke et al. This circuit contains the dimeric Broccoli aptamer placed within the three-way junction (3WJ) RNA motif, and this aptamer forms a secondary structure that is able to bind to a membrane-permeable, fluorescent dye known as DFHBI-1T. DFHBI-1T only fluoresces after binding to the Broccoli aptamer. This circuit places 3WJdb under the control of the constitutive P70a promoter.
Build: We acquired this circuit from Addgene (#87311).
Test: To test this circuit, we transformed it individually into BL21(DE3), which is the bacterial strain used in the original paper in which this circuit was constructed and tested (Alam et al., 2017). After growing a culture of transformed cells overnight (37°C and 250 RPM) and incubating with DFHBI-1T at room temperature for 45 minutes, we tested for aptamer fluorescence in our culture using a plate reader by comparing the fluorescence of the aptamer to samples of other transcriptional circuit sensors that were incubated with dye for the same amount of time, including the Split Broccoli system (Alam et al., 2017) and untransformed cells and LB (negative controls) and samples of our translational burden sensor circuit (positive control). The results of our test showed that 3WJ-Broccoli is able to bind to DFHBI-1T and produced a fluorescent signal in vivo. The next step of circuit testing was then to set up an experiment with 3WJ Broccoli and our test circuit, pBbB8k-csg-amylase, an arabinose-inducible curli fiber production circuit designed by researchers Birnbaum et al. In order to complete this step, we needed to do a cotransformation with the 3WJ-Broccoli circuit and our test circuit in E. coli BL21(DE3). After overnight incubation (37°C and 250 RPM), we made subcultures of our overnight cultures and grew all samples in plate reader plates. We compared the fluorescence of samples of 3WJ Broccoli individually transformed into the host, 3WJ Broccoli and our test circuit cotransformed into the host (uninduced), 3WJ Broccoli and our test circuit cotransformed into the host (induced with arabinose), untransformed BL21(DE3) cells, and LB. We measured the fluorescence of all overnight samples, all subcultures right before induction of the test circuit (T0), 1 hour after induction, 6 hours after induction, 12 hours after induction, 24 hours after induction, and 48 hours after induction. We repeated this experiment in plates a total of three times. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
Translational Burden Sensor Circuits
Design:For our translational burden sensor circuits, we tested two designs. The first version of this circuit, WM21_016, was designed by researchers Ceroni et al. (Ceroni et al., 2015) to test translational burden of genetic constructs within a cell. However, while researchers Ceroni et al. incorporated their circuit into the host genome, we utilized their circuit in plasmid form. Researchers Ceroni et al. used the strong constitutive promoter BBa_J23100 and an RBS designed (using an RBS calculator) specifically for codon optimized sfGFP. The sequence for codon optimized sfGFP was created for high expression levels using DNA2.0, and sfGFP was followed by a spacer and the synthetic terminator BBa_B1002. Based on Ceroni et al. circuit concept and design, our team designed WM21_013, a translational burden sensor consisting of the strong, constitutive Anderson promoter BBa_J23119, the RBS B0034 with a spacer included, the codon optimized sfGFP sequence used by researchers Ceroni et al. (2015), and the strong synthetic, bidirectional terminator BBa_B1006.
Build:In order to construct these circuits, we ordered both inserts (including promoter, RBS, codon-optimized sfGFP and terminator) flanked by unique nucleotide sequences (UNSs) 1 and 10, as gBlocks from IDT. We then used Gibson Assembly to join our insert with the vector pSB1C3 flanked by UNSs 1 and 10. Following Gibson Assembly, we transformed our translational burden sensor circuits individually into E. coli DH5-alpha using a standard heat shock method.
Test: To test the transcriptional sensor circuits, we transformed them individually into E. coli DH5-alpha. After growing a culture of transformed cells overnight (37°C and 250 RPM), we tested for sfGFP fluorescence using a plate reader by comparing the fluorescence of our circuits to untransformed cells and LB (negative controls) and samples of our translational burden sensor circuit (positive control). The results of our test showed that both circuits are functional and produce high levels of sfGFP. The next step of circuit testing was then to set up an experiment with our translational burden sensor circuits and our test circuit, pBbB8k-csg-amylase, an arabinose-inducible curli fiber production circuit designed by researchers Birnbaum et al. In order to complete this step, we needed to do a cotransformation with the translational burden sensor circuits and our test circuit in E. coli DH5-alpha. After overnight incubation (37°C and 250 RPM), we made subcultures of our overnight cultures and grew all samples in plate reader plates. We compared the fluorescence of samples of our translational burden sensor circuits individually transformed into the host, our translational burden sensor circuits and our test circuit cotransformed into the host (uninduced), our translational burden sensor circuits and our test circuit cotransformed into the host (induced with arabinose), untransformed E. coli DH5-alpha cells, and LB. We measured the fluorescence of all overnight samples, all subcultures right before induction of the test circuit (T0), 1 hour after induction, 6 hours after induction, 12 hours after induction, 24 hours after induction, and 48 hours after induction. We repeated these experiments in flasks a total of three times per translational burden sensor circuit. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
Post-Translational Modification Sensor Circuits
Phosphorylation Circuit
Design: Our team designed WM21_014 with the intent of quantifying differences in phosphorylation levels in host cells with and without an additional circuit. WM21_014 utilizes the EnvZ/OmpR osmoregulation system of E. coli to measure changes in phosphorylation via a fluorescence output (sfGFP). This circuit design is taken from Schmidl et al.
Build: In order to build this circuit, our team utilized the sequence in Schmidl et al. We ordered this sequence as a gene block from IDT DNA with UNS1 and UNS10 sequences flanking the gene block in order to insert it into the 1C3 plasmid backbone. Once our gene block arrived, we Gibsoned it into the appropriate backbone and transformed it into NEB5-alpha cells. To confirm the efficacy of our transformation, we performed a colony PCR using a forward primer of UNS1 and a reverse primer using UNS10. Once the PCR was run, we performed a gel electrophoresis on the product to confirm that the insert was the appropriate length. Our gel electrophoresis was able to confirm that all tested colonies contained the appropriate plasmid lengths. These confirmed colonies were grown overnight and converted into glycerol stocks, which were then used for the rest of the experiment.
Test: In order to test the functionality of WM21_014, our team performed a series of tests to determine fluorescence. As this system is supposed to fluoresce in response to changes in osmolarity, our team planned to grow this circuit in LB media with a 20% sucrose concentration overnight. After overnight growth, our team planned to measure the fluorescence of the culture with a plate reader. However, after our transformations, our team encountered an issue. Our colonies transformed with WM21_014 were fluorescent on the plate without having been induced. Obviously, our circuit was not working properly and our team decided to design a new circuit. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
Failure & Redesign: In response to the presence of fluorescence without induction of circuit WM21_014, our team designed WM21_015. This circuit is based off of Tokyo-NoKoGen’s 2012 circuit (BBa_K769001). However, instead of using GFP, our team used codon-optimized sfGFP (Ceroni et al., 2015). Our team used the same building and testing process, including the sequencing process, on WM21_015 as WM21_014 and was able to functionally confirm it.
Glycosylation Circuit
Design: We designed WM21_022 to quantify differences in glycosylation levels in host cells with or without an additional circuit. It makes use of the ftsLp2 promoter that initiates transcription of genes involved in glycosylation of peptidoglycan in E. coli, such as murG, a glycosyltransferase, and murC, a ligase that binds MurNAc to an amino acid (Barreteau et al, 2008). The promoter, which is regulated by the repressor LexA and was identified by Vicente et al, would assess glycosylation in the host cell via a fluorescence output by expressing sfGFP. The circuit featured strong RBS BBa_B0034 with a spacer (which had been successful with sfGFP expression in E. coli), codon optimized sfGFP, and strong terminator BBa_B1006. These parts were all used in the design of translation sensor circuit WM21_013 and had proven to be successful.
Build: In order to build this circuit, our team combined the sequence from the E. coli genome at the position indicated by Vicente et al., BBa_0034, sfGFP, and BBa_B1006. We ordered this sequence as a gene block from IDT DNA with UNS1 and UNS10 sequences flanking the gene block in order to insert it into the 1C3 plasmid backbone. Once our gene block arrived, we performed a Gibson assembly to insert the fragment into the 1C3 backbone and transformed it into NEB5-alpha cells. To confirm the efficacy of our transformation, we performed a colony PCR using a forward primer for UNS1 and a reverse primer for UNS10. Once the PCR was run, we performed a gel electrophoresis on the product to confirm that the insert was the appropriate length. Our gel electrophoresis was able to confirm that all tested colonies contained the appropriate plasmid lengths. These confirmed colonies were grown overnight and converted into glycerol stocks, which were then used for the rest of the experiment.
Test: Once we confirmed the transformation of the circuit, we tested its functionality by measuring its fluorescence compared to untransformed cells and successfully fluorescing cells. When these tests showed that the circuit was not fluorescing above the levels of the untransformed cells, we set about inducing the SOS response in the cells to derepress LexA from the circuit’s ftsLp2 promoter. The transformed cells were exposed to UV light and subinhibitory levels of Ciprofloxacin (Sassanfar et al, 1990, Baharoglu et al, 2011), but all attempts were unsuccessful at making induced, transformed cells consistently and significantly more fluorescent than untransformed cells. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
Failure: We designed this circuit with the goal of assessing another common mode of post translational modification besides phosphorylation in the host cells. Searching in the literature yielded no prior attempts to do this. This may be related to the fact that there is currently no characterization of protein glycosylation in non-pathogenic strains of E. coli. Glycosylation in E. coli is typically associated with toxicity to humans because glycosylated auto-transporter proteins aid in biofilm aggregation and binding to human cells (Sherlock et al, 2006). Knowing this, we decided our best option to represent glycosylation was the synthesis of peptidoglycan, so we selected the ftsLp2 promoter that initiates transcription of the mur operon, involved with peptidoglycan synthesis. However, we could find no prior use of this promoter in synthetic biology to design a circuit, so we were unsure if this design would be functional. The sequence was estimated by the location of LexA-binding site sequence homology analysis and S1 nuclease mapping analysis, but we believe it is most likely that the sequence we used for our design was incomplete or inaccurate.
Proteolysis Circuits
lonProtease
Design: We designed the lon sensor circuit, WM21_023, to report expression of the promoter which codes for the protease Lon in E. coli. This circuit places codon-optimized sfGFP after the promoter and the strong RBS BBa_B0034, allowing us to report expression via green fluorescence. The terminator BBa_B1002 is also used. Finally, between the RBS and sfGFP is a spacer which has been shown to allow for expression with BBa_B0034 and sfGFP (Clifton et al., 2018). Additionally, we flanked the circuit with the UNS 1 and UNS 10 sequences from Torella et al., 2014 in order to allow for easy Gibson assembly (Torella et al., 2014).
Build: After ordering our circuit from IDT as a gene block, we inserted it into the 1C3 plasmid backbone. We transformed the plasmid into NEB5-alpha cells, which we chose due to its high MiniPrep concentrations and success with single transformations and cotransformations with circuits in the past. We then confirmed the transformations by growing cells on plates with selection for chlor-resistance, which is conferred by the 1C3 backbone. Furthermore, we performed a colony PCR on four colonies and confirmed that they contained plasmids of the correct length using gel electrophoresis. All four bands were confirmed, and we made MiniPreps and corresponding glycerol stocks of cells from two of the colonies. The initial MiniPrep returned concentrations of below 50 ng/uL, but a second attempt gave a concentration of 89.4 ng/uL for Colony 1 and 62.5 ng/uL for Colony 2.
Test: We confirmed that the circuit produced green fluorescence by inoculating cells from our two glycerol stocks overnight and placing them in the plate reader along with two negative controls (LB alone and untransformed NEB5-alpha cells) and a positive control (Circuit 13, which we had previously seen to fluoresce). Although the positive control failed to fluoresce in this case for unknown reasons, cells from both colonies containing WM21_023 fluoresced far more than the negative controls. Additionally, the positive control did fluoresce during functional confirmation of the clpB and hslVU sensors; when compared to these fluorescence values, lon caused fluorescence that was about half as high as that of the positive control for both colonies. As a result, we decided to use our Colony 1 as our main source of cells, as its MiniPrep had the highest concentration of the two MiniPrepped colonies. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
hslVU Protease
Design: We designed the hslVU sensor circuit, Circuit 24, to report expression of the promoter which codes for the protease complex hslVU in E. coli. This circuit places codon-optimized sfGFP after the promoter and the strong RBS BBa_B0034, allowing us to report expression via green fluorescence. The terminator BBa_B1002 is also used. Finally, between the RBS and sfGFP is a spacer which has been shown to allow for expression with BBa_B0034 and sfGFP (Clifton et al., 2018). Additionally, we flanked the circuit with the UNS 1 and UNS 10 sequences from Torella et al., 2014 in order to allow for easy Gibson assembly (Torella et al., 2014).
Build: After ordering our circuit from IDT as a gene block, we inserted it into the 1C3 plasmid backbone. We transformed the plasmid into NEB5-alpha cells, which we chose due to its high MiniPrep concentrations and success with single transformations and cotransformations with circuits in the past. We then confirmed the transformations by growing cells on plates with selection for chlor-resistance, which is conferred by the 1C3 backbone. Furthermore, we performed a colony PCR on four colonies and confirmed that they contained plasmids of the correct length using gel electrophoresis. All four bands were confirmed, and we made MiniPreps and corresponding glycerol stocks of cells from two of the colonies. Cells from Colony 1 provided a MiniPrep concentration of 155.5, and cells from Colony 2 provided a concentration of 187.9, albeit with lower purity.
Test: We confirmed that the circuit produced green fluorescence by inoculating cells from our two glycerol stocks overnight and placing them in the plate reader along with two negative controls (LB alone and untransformed NEB5-alpha cells) and a positive control (Circuit 13, which we had previously seen to fluoresce). Cells from both colonies containing the hslVU sensor fluoresced at a similar level to the positive control. Additionally, we heat shocked an additional sample of cells from each colony to confirm the circuit’s sensitivity to some form of stress, as authors who have used the promoter in a circuit before have seen an increase in fluorescence after heat shock (Lien et al., 2009). We placed the cells, which were initially at 37ºC, into a 42ºC heating block for 20 minutes. We then measured green fluorescence in the plate reader and saw that, on average, Colony 1 induced cells fluoresced about 30% more than uninduced cells. Colony 2 induced cells fluoresced about 20% more. As a result, we decided to use our Colony 1 as our main source of cells; additionally, its MiniPrep had the highest concentration of the two MiniPrepped colonies. It is worth noting that if we had waited for a longer time after heat shock before measuring fluorescence, the increase would likely have been higher (Lien et al., 2004). This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
”Beyond Burden” Circuits
Heat Shock Response Proteins
dnaK and dnaJ (dnaKJ)Sensor
Design: With the goal of quantifying the changes in expression of both dnaK and dnaJ through fluorescence output, we designed a single sensor circuit, WM21_021, consisting of the constitutive dnaKJ operon promoter (Cowing et al.,1985), the sfGFP coding region, and the terminator BBa_B1006. To be able to do Gibson Assembly with ease, we added UNS1 and UNS10 sequences to the ends of our insert design before ordering. We also made sure to find multiple sources that used the same promoter sequence and tested its functionality in vivo. When ordering the circuit, we took advantage of the IDT design tools to confirm that IDT is able to produce these gene blocks.
Build: To build our plasmids, we first inserted the circuit using Gibson Assembly into the 1C3 plasmid backbone, which has chloramphenicol antibiotic resistance. We then transformed our plasmids into NEB5-alpha cells which we chose because of its high MiniPrep concentration yield and success with single transformations and cotransformations based on results from circuit WM21_015 (the phosphorylation circuit). To confirm that the cells had the plasmids with the right inserts, after selecting for the cells with the specific antibiotic resistance on the LB agar plates with chloramphenicol, we ran a colony PCR on 4 different single colonies from the successful overnight growth plate. After amplification of the inserts using colony PCR with a forward primer at UNS1 and a reverse primer at UNS10 (which encapsulated our insert), we then conducted gel electrophoresis to determine whether the inserted gene blocks were of the appropriate length according to what we had designed. For the WM21_021 circuit, which has a length of 1057 bp, all 4 colonies showed the correct bands close to the 1000 bp line. Three colonies were chosen for making MiniPreps and glycerol stocks with the last colony solution, made during colony PCR preparation, being put aside as a backup.
Test: To test whether the insert was functional, we needed to check if there was fluorescence production in cells with our circuit. Taking the heat-shocking protocol from Cha et al. (1999) as a model, we conducted a plate reader experiment to measure the fluorescence and OD600 levels of transformed cells that were heat shocked from 37 C to 42 C, cells that were not heat-shocked, untransformed NEB5-alpha cells (negative control), LB supplemented with chloramphenicol (negative control), and NEB5-alpha cells transformed with circuit WM21_013 which we had previously functionally confirmed to be producing green fluorescence constitutively (positive control). Interestingly, the heat shocked cells had fluorescence readings lower than those that were not heat-shocked. This was probably due to the adjustments we made to the model protocol by Cha et al. (1999) where they had originally been cold-shocked at 30 C and then taken to 42 C for the heat-shock for a longer time which may have been why they observed higher fluorescence for the heat-shocked cells in their results. Regardless, our results showed that the heat shocked cells and the non-heat shocked cells had relative fluorescence levels 5- to 10-fold higher than the controls, indicating that the dnaKJ operon promoter is indeed functional and allows for the expression of sfGFP. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
clpB (ATPase) Sensor
Design: We designed the clpB sensor circuit to report expression of the promoter which codes for the heat shock protein clpB in E. coli. Our initial design of this circuit, WM21_017, placed codon-optimized sfGFP after the promoter and the RBS which normally precedes clpB in E. coli, allowing us to report expression via green fluorescence. We included this RBS because the same was done by Kitagawa et al., 1991 (Kitagawa et al.,1991). The terminator BBa_B1002 was also used. Finally, between the RBS and sfGFP was a spacer which has been shown to allow for expression with BBa_B0034 and sfGFP (Clifton et al., 2018). Additionally, we flanked the circuit with the UNS 1 and UNS 10 sequences from Torella et al., 2014 in order to allow for easy Gibson assembly (Torella et al., 2014).
Build: After ordering our circuit from IDT as a gene block, we inserted it into the 1C3 plasmid backbone. We transformed the plasmid into NEB5-alpha cells, which we chose due to its high MiniPrep concentrations and success with single transformations and cotransformations with circuits in the past. We then confirmed the transformations by growing cells on plates with selection for chlor-resistance, which is conferred by the 1C3 backbone. Furthermore, we performed a colony PCR on four colonies and confirmed that they contained plasmids of the correct length using gel electrophoresis. All four bands were confirmed, and we made MiniPreps and corresponding glycerol stocks of cells from two colonies. Cells from Colony 1 provided a MiniPrep concentration of 103.7, and cells from Colony 2 provided a concentration of 192.3, albeit with lower purity.
Test: We confirmed that the circuit produced green fluorescence by inoculating cells from our two glycerol stocks overnight and placing them in the plate reader along with two negative controls (LB alone and untransformed NEB5-alpha cells) and a positive control (Circuit 13, which we had previously seen to fluoresce). Cells from both colonies containing WM21_017 fluoresced about half as much as the positive control, which was not as high as we had hoped. Additionally, we heat shocked an additional sample of cells from each colony to confirm the circuit’s sensitivity to some form of stress, as authors who have used the promoter in a circuit before have seen an increase in fluorescence after heat shock (Cha et al., 1999). We placed the cells, which were initially at 37ºC, into a 42ºC heating block for 20 minutes. We then measured green fluorescence in the plate reader and saw that, on average, Colony 1 induced cells fluoresced about 10% more than uninduced cells. Colony 2 induced cells fluoresced about 60% more. It is worth noting that if we had waited for a longer time after heat shock before measuring fluorescence, the increase would likely have been higher (Cha et al., 1999).
Additionally, when we cotransformed WM21_017 with pBbB8k-csg-amylase into NEB5-alpha cells, we saw that fluorescence was consistently higher than when WM21_017 was transformed alone into cells. We had designed this circuit to show increased fluorescence when cotransformed with a second circuit such as pBbB8k-csg-amylase. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
Failure & Redesign: Although it was not a failure, we hoped to improve upon the fluorescence of WM21_017, which was only half as fluorescent as Circuit 13. More importantly, WM21_017 did not succeed at producing higher fluorescence when cotransformed alongside another circuit. Fortunately, we had considered that WM21_017 might have produced lower fluorescence than was possible, and therefore designed a different version of the clpB sensor circuit which includes the RBS BBa_B0034 rather than the RBS native to E. coli. This design, WM21_018, is more in line with our Circuit 13, which successfully produced fluorescence and includes BBa_B0034.
Build: We built this circuit in the same way as WM21_017, transforming it into cells and confirming that plasmids of the correct length were present through colony PCR and gel electrophoresis. The bands for Colonies 3 and 4 looked the most clearly confirmed, so we made MiniPreps and corresponding glycerol stocks of cells from those colonies. Cells from Colony 3 provided a MiniPrep concentration of 212.3, while cells from Colony 4 provided a concentration of 181.1. We chose to use cells from Colony 3 due to the higher MiniPrep concentration.
Test: We functionally confirmed WM21_018 in the same way as was used for WM21_017. Cells from both colonies containing WM21_018 fluoresced over twice as much as the positive control before heat shock, indicating an improvement from WM21_017. After heat shock, cells from Colony 3 did not fluoresce any more than non-heat-shocked cells. Although we had hoped that heat shock would cause an increase in fluorescence, we believed that if we had measured fluorescence after a longer time had elapsed after heat shock, there would be a larger increase, as this would have more closely reflected the methods used by Cha et al., 1999 to observe an increase in fluorescence (Cha et al., 1999).
When we cotransformed WM21_018 with pBbB8k-csg-amylase into NEB5-alpha cells, we did see that fluorescence was higher over time for cotransformed cells than cells with the circuit alone. This suggests that, unlike WM21_017, WM21_018 successfully responds to the presence of pBbB8k-csg-amylase with increased sfGFP expression.
Secretion (AG43) Sensors
Design: We designed two circuits using two different promoters for the flu gene (flup1 and flup2) with the intent to sense cellular secretion as a response to the insertion of a circuit.
Build: We then built these circuits using Gibson Assembly after ordering a gene block for each circuit that was bookended by UNS1 and UNS10 for easy insertion into the 1C3 backbone. After transformation into NEB5-alpha cells, we proceeded to run a colony PCR, where we selected 4 colonies per circuit to verify. After amplification of the inserts using colony PCR with a forward primer at UNS1 and a reverse primer at UNS10 (which encapsulated our insert), we then conducted gel electrophoresis to determine whether the inserted gene blocks were of the appropriate length according to what we had designed. After verifying the length of the gene blocks, we then inoculated using the PCR tubes and let the cultures grow overnight. The next day, we conducted MiniPreps to create working -20ºC and -80ºC glycerol stocks for each of our plasmids.
Test: To test the functionality of our circuits, we inoculated from our -20ºC glycerol stocks and let the cultures grow overnight. We then pipetted three wells, each containing 200 µL of our culture, into a 96-well plate, along with three wells of the same volume of each of our positive and negative controls. Our positive control was WM21_013, a circuit that we had already found to exhibit incredibly high fluorescence values, and our negative control was LB, as the medium we used should not have been fluorescent. We then ran the plate through a Synergy H-1 plate reader, and determined whether the wells containing our plasmid cultures were more fluorescent than the negative control, with the positive control as a benchmark for the expected high level of fluorescence given our use of sfGFP. This part was successfully sequenced by Epoch Life Science and sequence-confirmed using Benchling tools.
Failure & Redesign: Both of the secretion circuits failed to be functionally confirmed. We hypothesize that this may be due to the fact that the promoters we used were not well-characterized, given that the rest of these circuits were identical to the corresponding parts of other functional circuits we had tested. If we were to redesign this circuit in the future, it would be prudent to find a promoter for the flu gene that has been better characterized, although it is possible that such a promoter does not currently exist in the literature.
*Our team understands that many of these circuits can fit into both the Central Dogma sensor circuit category and "Beyond Burden" sensor circuit category; therefore, these circuits were grouped together for clarity.
References
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