Part:BBa_K4387000

Nitric Oxide Sensing Promoter pNorVβ

Usage and Biology

The inducible pNorVβ is an optimized nitric oxide sensitive promoter regulated by NorR. The corresponding integration host factor IHF2 binding site was removed from the promoter pNorV to exhibit good sensitivity and dosage response at a low range of inducer DETA/NO, the used nitric oxide source in our experiments, activating the downstream genes' transcription [1]. In our constructs below, this promoter was coupled to a superfolder GFP and the transcriptional regulator NorR BBa_K4387001 which creates a feedback loop to finetune the amount of NorR to the number of free NO molecules. It is also an improved part of the 2016 ETH iGEM team pNorV promoter.

This promoter was tested in the bacterial strain E.coli Nissle 1917.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization and Measurements

Measuring parts with different approaches and comparing them to provide a more insightful and multilayered characterization is essential in Synthetic Biology. Here, we focused on two methods:

(i) time-lapse plate reader assays to measure the sensitivity of our circuit to NorR in a dynamic manner and under different concentrations of inducer; and

(ii) endpoint flow cytometry assays to measure the behavior of our circuits at the single-cell scale.

With the first assay, we uncovered essential kinetic information about the circuits on the populational level (every measurement is an average of the individual expression patterns in the sample). With the second assay, we delved deeper into the cell populations to characterize other essential properties of our system, such as expression noise and dose-dependent responses to different inducer concentrations.

To make our experiments reproducible during plate reader assays (PHERAstar FSX - λEx: 485 nm, λEm: 530 nm), we measured each sample for 16 hours at 37°C and constant orbital shaking, using three biological replicates (three individual colonies per circuit) and three technical replicates (three wells per biological replicate).

We performed the data analysis as follows:

1. Subtracted blanks from raw data
2. Normalize GFP by OD600
3. Calculate technical means of GFP/OD600 normalized data
4. Calculate the biological means of GFP/OD600 normalized data
5. Calculate the biological standard deviation of GFP/OD600 normalized data
6. When necessary, perform imputation. Usually, the first normalized measurements are noisy and unreliable as OD600 values can be very low and significantly impact normalization. Thus, when individual normalized values are extremely high or low (sometimes negative due to blank correction), imputation was used following a na_kalman() function from the ImputeTS R package.
7. Additional transformations, such as log transformations.

Hence, our plots show the averages and standard deviations for the biological replicates for each sample for each time point.

For the flow cytometry experiment, cell cultures were grown overnight in LB medium supplemented with antibiotic, diluted in 2mL of M9 (supplemented with glucose, cas amino acids and an antibiotic) in a 1:10 ratio (v/v), induced with different NO concentrations and grown for 7 hours in a shaker (37C, 220 RPM). Samples were then chilled on ice to halt cell growth and diluted in 1mL of cold PBS (1:500 v/v ratio). A total of 100,000 cells per sample was measured in a BD FACSCanto II flow cytometer (FSC: 625V, SSC: 420V, FITC: 650V, Event threshold: FSC & SSC > 200, Channel: FITC (λEx 488 nm / λEm. 530/30 nm, High flow rate: ~ 10,000 events/s).

We performed all analyses using in-house R scripts.

Nitric Oxide Sensing Genetic Circuit With One Ribosomal Binding Site

This part consists of the inducible pNorVβ promoter, a superfolder GFP preceded by one strong ribosomal binding site (BBa_B0034), the NorR regulator, and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR based on the amount of nitric oxide present [1]. The presence of nitric oxide would activate pNorVβ to induce GFP and NorR expression. Thereby, we ensure that high amounts of NorR will be produced only when NO is present.

Time-Lapse Plate Reader Assay

Figure 3a: Response of the Genetic Circuit pNorVβ including 1 RBS to DETA/NO induction. We observe that the GFP expression is ~3000 GFP/OD for NO-concentration of 2mM. Comparing this construct with the others in figure 3b and 3c, we could prove that pNorVβ is more powerful than the ETH promoter but less effective than the constructs with additional ribosomal binding sites.

To make our experiments reproducible during plate reader assays (PHERAstar FSX - λEx: 485 nm, λEm: 530 nm), we measured each sample for 16 hours at 37°C and constant orbital shaking, using three biological replicates (three individual colonies per circuit) and three technical replicates (three wells per biological replicate).

We performed the data analysis as follows:

1. Subtracted blanks from raw data
2. Normalize GFP by OD600
3. Calculate technical means of GFP/OD600 normalized data
4. Calculate the biological means of GFP/OD600 normalized data
5. Calculate the biological standard deviation of GFP/OD600 normalized data
6. When necessary, perform imputation. Usually, the first normalized measurements are noisy and unreliable as OD600 values can be very low and significantly impact normalization. Thus, when individual normalized values are extremely high or low (sometimes negative due to blank correction), imputation was used following a na_kalman() function from the ImputeTS R package.
7. Additional transformations, such as log transformations.

Hence, our plots show the averages and standard deviations for the biological replicates for each sample for each time point.

Figure 3b: Response of Different Gene Circuits to DETA/NO Induction. Induction of plasmids BBa_K4387003 to BBa_K4387007. For each plasmid, it plots the emission of GFP over time, after induction with 6 different DETA/NO concentrations. Construct pNorVβ including 2 RBS has the highest GFP emission, whereas pNorV of the 2016 ETH team has the lowest GFP emission.

Figure 3c: Dose Response of all Constructs to DETA/NO. Plotted are the highest GFP expression levels under different concentrations of DETA/NO

Because the standard deviations overlap, we thought that we might be able to reduce the standard deviations and get a clearer result if we had more samples. To get more samples, we also performed a flow cytometer.

Endpoint Flow Cytometry Assay

For the flow cytometry experiment, cell cultures were grown overnight in LB medium supplemented with antibiotic, diluted in 2mL of M9 (supplemented with glucose, cas amino acids, and an antibiotic) in a 1:10 ratio (v/v), induced with different NO concentrations and grown for 7 hours in a shaker (37C, 220 RPM). Samples were then chilled on ice to halt cell growth and diluted in 1mL of cold PBS (1:500 v/v ratio). A total of 100,000 cells per sample was measured in a BD FACSCanto II flow cytometer (FSC: 625V, SSC: 420V, FITC: 650V, Event threshold: FSC & SSC > 200, Channel: FITC (λEx 488 nm / λEm. 530/30 nm, High flow rate: ~ 10,000 events/s).

Figure 4a: Violin Plots and Boxplots for Our 6 Different Constructs. These plots represent the distribution of FITC-H (green fluorescence) values for our six different plasmids at four different concentrations of DETA/NO. Each measurement consisted of 100'000 cells.

Figure 4b: Noise Levels of Our 6 Different Constructs. Noise levels are measured as the coefficient of variation for construct sample and NO concentration. Most constructs show low noise levels at high concentrations.

While figure 4a shows that the construct with pNorVβ and 2 RBS (BBa_K4387006) has higher overall GFP expression values, it is also leakier than this construct with pNorVβ and 1 RBS (BBa_K4387005) or the construct with pNorVβ and 3 RBS (BBa_K4387007). If high GFP expression is required, but some leakiness does not matter much, we recommend choosing BBa_K4387006. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts BBa_K4387005 or BBa_K4387007 instead.

Nitric Oxide Sensing Genetic Circuit With Two Ribosomal Binding Sites

In the frame of our project, we wanted to improve the construct BBa_K4387005 by adding one more ribosomal binding site to see if we could achieve a higher GFP response.

Thus this part consists of the inducible pNorVβ promoter, a superfolder GFP preceded by two strong ribosomal binding sites (BBa_B0029, BBa_B0034), the (NorR regulator), and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR based on the amount of nitric oxide present [1]. The presence of nitric oxide would activate pNorVβ to induce GFP and NorR expression. Thereby, we ensure that high amounts of NorR will be produced only when NO is present.

Time-Lapse Plate Reader Assay

Figure 5a: Response of the Genetic Circuit pNorVβ including 2 RBS to DETA/NO induction. We observe that the GFP expression is ~20'000 GFP/OD for NO-concentration of 2mM. Comparing this construct with the others in figure 5b and 5c, we could prove that this genetic circuit with pNorVβ including 2 RBS is the most effective construct between all six.

To make our experiments reproducible, during plate reader assays (PHERAstar FSX - λEx: 485 nm, λEm: 530 nm), we measured each sample for 16 hours at 37°C and constant orbital shaking, using three biological replicates (three individual colonies per circuit) and three technical replicates (three wells per biological replicate).

We performed the data analysis as follows:

1. Subtracted blanks from raw data
2. Normalize GFP by OD600
3. Calculate technical means of GFP/OD600 normalized data
4. Calculate the biological means of GFP/OD600 normalized data
5. Calculate the biological standard deviation of GFP/OD600 normalized data
6. When necessary, perform imputation. Usually, the first normalized measurements are noisy and unreliable as OD600 values can be very low and significantly impact normalization. Thus, when individual normalized values are extremely high or low (sometimes negative due to blank correction), imputation was used following a na_kalman() function from the ImputeTS R package.
7. Additional transformations, such as log transformations.

Hence, our plots show the averages and standard deviations for the biological replicates for each sample for each time point.

Figure 5b: Response of Different Gene Circuits to DETA/NO Induction. Induction of plasmids BBa_K4387003 to BBa_K4387007. For each plasmid, it plots the emission of GFP over time, after induction with 6 different DETA/NO concentrations. Construct pNorVβ including 2 RBS has the highest GFP emission, whereas pNorV of the 2016 ETH team has the lowest GFP emission.

Figure 5c: Dose Response of all Constructs to DETA/NO. Plotted are the highest GFP expression levels under different concentrations of DETA/NO

Because the standard deviations overlap, we thought that we might be able to reduce the standard deviations and get a clearer result if we had more samples. To get more samples, we also performed a flow cytometry.

Endpoint Flow Cytometry Assay

For the flow cytometry experiment, cell cultures were grown overnight in LB medium supplemented with antibiotic, diluted in 2mL of M9 (supplemented with glucose, cas amino acids, and an antibiotic) in a 1:10 ratio (v/v), induced with different NO concentrations and grown for 7 hours in a shaker (37C, 220 RPM). Samples were then chilled on ice to halt cell growth and diluted in 1mL of cold PBS (1:500 v/v ratio). A total of 100,000 cells per sample was measured in a BD FACSCanto II flow cytometer (FSC: 625V, SSC: 420V, FITC: 650V, Event threshold: FSC & SSC > 200, Channel: FITC (λEx 488 nm / λEm. 530/30 nm, High flow rate: ~ 10,000 events/s).

Figure 6a: Violin Plots and Boxplots for Our 6 Different Constructs. These plots represent the distribution of FITC-H (green fluorescence) values for our six different plasmids at four different concentrations of DETA/NO. Each measurement consisted of 100'000 cells.

Figure 6b: Noise Levels of Our 6 Different Constructs. Noise levels are measured as the coefficient of variation for construct sample and NO concentration. Most constructs show low noise levels at high concentrations.

While figure 6a shows that the construct with pNorVβ and 2 RBS (BBa_K4387006) has higher overall GFP expression values, it is also leakier than the construct with pNorVβ and 1 RBS (BBa_K4387005) or the construct with pNorVβ and 3 RBS (BBa_K4387007). If high GFP expression is required, but some leakiness does not matter much, we recommend choosing BBa_K4387006. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts BBa_K4387005 or BBa_K4387007 instead.

Nitric Oxide Sensing Genetic Circuit With Three Ribosomal Binding Sites

In the frame of our project, we wanted to further improve the construct BBa_K4387005 by adding two more ribosomal binding sites to see if we could achieve a higher GFP response.

Thus this part consists of the inducible pNorVβ promoter, superfolder GFP preceded by three strong ribosomal binding sites (BBa_K4387020 [2], BBa_B0029, BBa_B0034), the NorR regulator, and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part. Due to the competitive binding of the activated and inactivated NorR on the promoter, we decided on this construct with a positive feedback loop that adjusted the levels of NorR based on the amount of nitric oxide present [1]. The presence of nitric oxide would activate pNorVβ to induce GFP and NorR expression. Thereby, we ensure that high amounts of NorR will be produced only when NO is present.

Time-Lapse Plate Reader Assay

Figure 7a: Response of the Genetic Circuit pNorVβ including 3 RBS to DETA/NO induction. We observe that the GFP expression is ~9000 GFP/OD for NO-concentration of 2mM. Comparing this construct with the others in figure 7b and 7c, we could prove that this genetic circuit with pNorVβ including 3 RBS is more powerful than the ETH promoter and the genetic circuit with pNorVβ including 1 RBS, but less effective than the construct with pNorVβ including 2 RBS.

To make our experiments reproducible during plate reader assays (PHERAstar FSX - λEx: 485 nm, λEm: 530 nm), we measured each sample for 16 hours at 37°C and constant orbital shaking, using three biological replicates (three individual colonies per circuit) and three technical replicates (three wells per biological replicate).

We performed the data analysis as follows:

1. Subtracted blanks from raw data
2. Normalize GFP by OD600
3. Calculate technical means of GFP/OD600 normalized data
4. Calculate the biological means of GFP/OD600 normalized data
5. Calculate the biological standard deviation of GFP/OD600 normalized data
6. When necessary, perform imputation. Usually, the first normalized measurements are noisy and unreliable as OD600 values can be very low and significantly impact normalization. Thus, when individual normalized values are extremely high or low (sometimes negative due to blank correction), imputation was used following a na_kalman() function from the ImputeTS R package.
7. Additional transformations, such as log transformations.

Hence, our plots show the averages and standard deviations for the biological replicates for each sample for each time point.

Figure 7b: Response of Different Gene Circuits to DETA/NO Induction. Induction of plasmids BBa_K4387003 to BBa_K4387007. For each plasmid, it plots the emission of GFP over time, after induction with 6 different DETA/NO concentrations. Construct pNorVβ including 2 RBS has the highest GFP emission, whereas pNorV of the 2016 ETH team has the lowest GFP emission.

Figure 7c: Dose Response of all Constructs to DETA/NO. Plotted are the highest GFP expression levels under different concentrations of DETA/NO

Because the standard deviations overlap, we thought that we might be able to reduce the standard deviations and get a clearer result if we had more samples. To get more samples, we also performed a flow cytometry.

Endpoint Flow Cytometry Assay

For the flow cytometry experiment, cell cultures were grown overnight in LB medium supplemented with antibiotic, diluted in 2mL of M9 (supplemented with glucose, cas amino acids and an antibiotic) in a 1:10 ratio (v/v), induced with different NO concentrations and grown for 7 hours in a shaker (37C, 220 RPM). Samples were then chilled on ice to halt cell growth and diluted in 1mL of cold PBS (1:500 v/v ratio). A total of 100,000 cells per sample was measured in a BD FACSCanto II flow cytometer (FSC: 625V, SSC: 420V, FITC: 650V, Event threshold: FSC & SSC > 200, Channel: FITC (λEx 488 nm / λEm. 530/30 nm, High flow rate: ~ 10,000 events/s).

Figure 8a: Violin Plots and Boxplots for Our 6 Different Constructs. These plots represent the distribution of FITC-H (green fluorescence) values for our six different plasmids at four different concentrations of DETA/NO. Each measurement consisted of 100'000 cells.

Figure 8b: Noise Levels of Our 6 Different Constructs. Noise levels are measured as the coefficient of variation for construct sample and NO concentration. Most constructs show low noise levels at high concentrations.

While figure 8a shows that the construct with pNorVβ and 2 RBS (BBa_K4387006) has higher overall GFP expression values, it is also leakier than the construct with pNorVβ and 1 RBS (BBa_K4387005) or this construct with pNorVβ and 3 RBS (BBa_K4387007). If high GFP expression is required, but some leakiness does not matter much, we recommend choosing BBa_K4387006. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts BBa_K4387005 or BBa_K4387007 instead.

Nitric Oxide Sensing Genetic Circuit Without the NorR regulator

In the frame of our project, we wanted to improve the sensitivity of our construct BBa_K4387005. For this purpose, we removed the codon-optimized NorR, creating a circuit that would rely on endogenous NorR.

Thus this part consists of the inducible pNorVβ promoter, superfolder GFP preceded by two strong ribosomal binding sites (BBa_B0029, BBa_B0034), and a double forward terminator. We chose a high-copy backbone from Twist Bioscience for this part.

Time-Lapse Plate Reader Assay

Figure 9: Induction Response to DETA/NO for the Genetic Circuits pNorVβ with 2 RBS, with and without NorR We observe that the GFP expression is ~20'000 GFP/OD for NO-concentration of 2mM for the construct with NorR, while the GFP expression is ~13'000 GFP/OD for NO-concentration of 2mM for the construct without NorR. Comparing these constructs proves that removing the codon-optimized transcriptional regulator NorR did not improve the NO-sensing range. Instead, it reduced the GFP expression at the same NO concentration.

To make our experiments reproducible during plate reader assays (PHERAstar FSX - λEx: 485 nm, λEm: 530 nm), we measured each sample for 16 hours at 37°C and constant orbital shaking, using three biological replicates (three individual colonies per circuit) and three technical replicates (three wells per biological replicate).

We performed the data analysis as follows:

1. Subtracted blanks from raw data
2. Normalize GFP by OD600
3. Calculate technical means of GFP/OD600 normalized data
4. Calculate the biological means of GFP/OD600 normalized data
5. Calculate the biological standard deviation of GFP/OD600 normalized data
6. When necessary, perform imputation. Usually, the first normalized measurements are noisy and unreliable as OD600 values can be very low and significantly impact normalization. Thus, when individual normalized values are extremely high or low (sometimes negative due to blank correction), imputation was used following a na_kalman() function from the ImputeTS R package.
7. Additional transformations, such as log transformations.

Hence, our plot shows the averages and standard deviations for the biological replicates for each sample for each time point.

According to figure 9, removing the codon-optimized transcriptional regulator NorR did not improve the NO-sensing range. Instead, it reduced the GFP expression at the same NO concentration as in previous experiments.

Endpoint Flow Cytometry Assay

For the flow cytometry experiment, cell cultures were grown overnight in LB medium supplemented with antibiotic, diluted in 2mL of M9 (supplemented with glucose, cas amino acids, and an antibiotic) in a 1:10 ratio (v/v), induced with different NO concentrations and grown for 7 hours in a shaker (37C, 220 RPM). Samples were then chilled on ice to halt cell growth and diluted in 1mL of cold PBS (1:500 v/v ratio). A total of 100,000 cells per sample was measured in a BD FACSCanto II flow cytometer (FSC: 625V, SSC: 420V, FITC: 650V, Event threshold: FSC & SSC > 200, Channel: FITC (λEx 488 nm / λEm. 530/30 nm, High flow rate: ~ 10,000 events/s).

Figure 10a: Violin Plots and Boxplots for Our 6 Different Constructs. These plots represent the distribution of FITC-H (green fluorescence) values for our six different plasmids at four different concentrations of DETA/NO. Each measurement consisted of 100'000 cells.

Figure 10b: Noise Levels of Our 6 Different Constructs. Noise levels are measured as the coefficient of variation for construct sample and NO concentration. Most constructs show low noise levels at high concentrations.

As shown in the violin plots, the circuit without NorR, although it is not leakier than the other constructs, has a relatively high noise level (figures 10a and 10b). Since its response to the induction with DETA/NO is also decreased compared to its counterpart with the feedback loop, we consider this construct inadequately suited as a nitric oxide sensor (figure 10a).

While figure 10a shows that the construct with pNorVβ and 2 RBS (BBa_K4387006) has higher overall GFP expression values, it is also leakier than the construct with pNorVβ and 1 RBS (BBa_K4387005) or the construct with pNorVβ and 3 RBS (BBa_K4387007). If high GFP expression is required, but some leakiness does not matter much, we recommend choosing BBa_K4387006. If lower leakiness is essential, but GFP expression does not need to be very high, we recommend using parts BBa_K4387005 or BBa_K4387007 instead.

Exchanging the sfGFP with a Single Domain Antibody

This composite part contains the nitric oxide sensor pNorVß (BBa_K4387000), followed by 2 different ribosome binding sites. The promoter induces upon NO binding the expression of a monovalent anti-TNFα nanobodoy BBa_K4387996. The additional NorR at the end of the composite part enhances the positive feedback-loop, increasing the response of the promoter to NO. [1] Together with the hemolysin A secretion machinery BBa_K4387987, a complete genetic circuit is obtained that allows secretion of nanobodies [3] or if exchanged, secretion of other proteins of interest.

Western Blot

Figure 1: Double transformed E. coli Nissle 1917, able to secrete the monovalent nanobody VHH#2B, were induced with different DETA/NO concentrations (0mM, 1mM and 2mM) and incubated at 37°C overnight. Anti-myc antibodies were used in this Western blot to stain secreted nanobodies in the bacterial supernatant.

We double transformed our chassis, the probiotic E. coli Nissle 1917, with the high copy plasmid containing this composite part required for induced nanobody expression, and the medium copy number plasmid containing the composite part BBa_K4387987 needed for the secretion system. Liquid overnight cultures of transformed bacteria were grown and induced by adding 2mM, 1mM or 0mM NO respectively to the cultures. DETA/NO was used as a nitric oxide source for the induction experiments. On the next day, the cells were centrifuged, and the supernatant was run on a gel. To see if nanobodies of the correct size have been secreted by the bacteria, we conduct a Western blot by detecting the myc-tag fused to the nanobodies (Figure 1).

As seen in figure 1, we received a band with the size of approximately 45 kDa which fits the expected size of the monovalent nanobody candidate VHH#2B together with the myc-tag and HlyA-tag. We can therefore assume that the bacteria were able to secrete whole nanobodies.

However, the first two bands showing the bacterial samples that have not been induced with DETA/NO and therefore should not have secreted nanobodies are visible, indicating that the promoter is leaky. To investigate further, we compared the intensity of the bands that we received from the Western blot with imageJ. For each condition a numerical average was calculated:

image J values

DETA/NO	Band 1	Band 2	Average
0 mM	6296.811	4532.326	5414.5686
1 mM	5664.619	6698.811	6181.715
2 mM	6987.589	8298.468	7643.0285

On average the bands from the 2mM DETA/NO are 41% more intense than the control indicating an increased protein secretion upon DETA/NO induction. However, the non-induced expression appears to be quite leaky. A possible explanation for the leakiness might be the two ribosomal binding sites that follow the promoter, leading to an enhance promoter activity but also to more leakiness. Additionally, the bacterial cultures were grown overnight for about 15 hours at 37°C, leading to a dense E. coli culture. It is possible that over time nitric oxide might have been metabolically produced by the bacteria and accumulated, leading to an increasing self-induction over this long period of time.

ELISA

Figure 2: ELISA testing TNFα-binding capabilities of the secreted monovalent nanobody VHH#2B obtained from E. coli Nissle 1917 after nitric oxide induction, compared to purified and secreted nanobodies form MC1061

To prove that the secreted nanobodies not only have the correct size but are also able to elicit their TNFα-binding abilities, we performed an ELISA (Figure 2). Adalimumab, a monoclonal anti-TNFα antibody already used in the clinics to treat IBD patients, served as a positive control (wells C1-2), and a sybody against a membrane protein was the negative control (wells C3-4). We could show that the transformed E. coli Nissle 1917 is able to secrete functional anti-TNFα nanobodies upon nitric oxide induction (row A).

References

• [1] Xiaoyu J. Chen, Baojun Wang, Ian P. Thompson, and Wei E. Huang et al. Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells ACS Synthetic Biology 2021 10 (10), 2566-2578 DOI: 10.1021/acssynbio.1c00223
• [2] Ayelet Levin-Karp, Uri Barenholz, Tasneem Bareia, Michal Dayagi, Lior Zelcbuch, Niv Antonovsky, Elad Noor, and Ron Milo et al. Quantifying Translational Coupling in E.coli Synthetic Operons Using RBS Modulation and Fluorescent Reporters ACS Synthetic Biology 2013 2 (6), 327-336 DOI: 10.1021/sb400002n
• [3] Silence, Karen, Lauwereys, Marc, De Haard, Hans, et al. "Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor", Int. Publication Number: WO 2004/041862 A2, 21 May 2004