Experiments

Anti-TNFα nanobodies and secretion system:

For the characterization of the nanobody binding capabilities, we mainly focused on two different methods:

  • (i) testing TNFα-binding with an Enzyme-linked Immunosorbent Assay (ELISA)
  • (ii) characterizing immune response of human monocytes to TNFα actions

The ELISA works in a step-wise manner. First, we coated a 96-well plate with protein A, which serves as an anchor for the anti-myc antibody. The anti-myc antibody binds the myc tag fused to our nanobody constructs. Added biotinylated TNFα will be bound by the nanobodies if they are correctly formed. Finally, successful binding of TNFα is made visible by adding developing buffer to streptavidin-peroxidase bound to the biotin of TNFα. The absorbance of each well is then measured at 650 nm in a plate reader. With the second method we investigated the effect of TNFα on human monocytes and by adding our nanobodies, checked their ability to counteract the pro-inflammatory signalling by inhibiting TNFα through successful binding.

The secretion of nanobodies by E. coli with the hemolysin A secretion system is a vital part of our project. To obtain reproducible results we followed the same procedure for our secretion experiments:

  • Check the bacterial supernatant for nanobodies with a Western blot
  • Test their proper folding and binding with an ELISA

The myc-tag fused to the nanobodies was once again key in detecting secreted nanobodies. We ran the bacterial supernatant of overnight cultures after induction on a native PAGE gel and then transferred the proteins to the western blot membrane. Afterwards, by incubating the membrane with primary anti-myc antibodies and secondary antibodies we were able to visualise the nanobody bands by imaging the chemiluminescence. This confirmed their presence and gave information on the secretion intensity. The ELISA was performed as described above. Instead of purified nanobodies, the bacterial supernatant was used to show the proper binding capabilities of secreted nanobodies.

Nitric oxide sensing circuit:

Measuring parts with different approaches to provide a more insightful 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 DETA/NO in a dynamic manner.
  • (ii) endpoint flow cytometry assays to measure the behaviour of our circuits at the single-cell scale.

With the first assay, we uncovered essential kinetic information about the circuits on the population 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. Subtract 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 (37°C, 220 RPM). Samples were then chilled in 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.