Team:Aboa/Measurement
Measurement
This page contains a summary of all the measurement methods that were used in the Team Aboa iGEM project. We measured different promoters' effect on production rates, antibody activities, concentrations and thermal stability.
Product optimization
Before starting to produce antibodies with E.coli 321.A -strain, its capability of producing proteins needed to be characterized and tested to see how different promoters affected its production rate.
GFP-TAG-RFP production optimization
The GFP-TAG-RFP's function was measured in a 96-well plate with Cytation 5 (BioTek, Vinooski, VT, USA). The effects of three parameters were studied: concentration of IPTG, arabinose and p-azido-l-phenylalanine. We measured the signals using fluorescence channels of preset GFP (excitation: 395 nm, emission: 509 nm) and mCherry (excitation 570 nm, emission: 615 nm) programs. Measurements were performed overnight in 30 minute intervals. The change of fluorescence ratio of RFP and GFP was used as a way to normalize signals as the pUC19 lac promoter was leaky and we didn’t want to measure anything expressed before induction. This relative change could be used to study the effectiveness of pAzF incorporation and the general properties of this biobrick when it is expressed using expanded genetic code and our GRO cell strain.
Fab product optimization
We used Goat Anti Human plates and a 2A11-Eu as described in Experiments to detect Fab activities from a production optimization experiment ran on a 96-well plate. We used Cytation 5 to culture the cells also as explained in GFP-TAG-RFP production experiments.
GFP-TAG-RFP production optimization
With our test setting we were actually able to detect previously undocumented RFP from the reporter. The relative change in GFP and RFP fluorescent signal changes were further compared and they are represented in a figure below. From the figure 3 it can be concluded, that our system was leaky and that in the absence or at low concentrations of pAzF and arabinose, there might be something else incorporating into the fusion protein. However, the relative signal ratio was pretty much the same at 0.5 % and 1 % of arabinose, which is probably the point, where the pAzF-tRNA production is optimal.
Figure 1. The relative change of fluorescence measured in preset mCherry and GFP channels. pAzF has the strongest effect on the expression of RFP when the synthetase is present. This also shows that when the synthetase is not present, there still is some misincorporation of other amino acids to the TAG site as it cannot be terminated either. IPTG does not have an effect on the reading of TAG stop codon as expected.
Fab product optimization
Figure 2. Production optimization of Fab variants. The cells were tested (A and B) without pAzF present (C and D) and 1 mM pAzF present. (A and C) Concentration of Arabinose was tested and (B and D) concentration of IPTG was tested. From here we can observe that pAzF has the most dramatic effect in expression of the Fab when the synthetase is present. IPTG has a relative small, if any effect on the expression which is somewhat surpricing. We chose to use 1mM pAzF, 0,5 mM IPTG and 0,5 % Arabinose in our work. This also proved that we can produce Fabs using our GRO strain.
Antibody activity assay
Once antibodies were purified, they needed to be analyzed to ensure they really were what they were supposed to be, humanized Fabs. This was confirmed with antibody activity immunoassay.
To detect the Fabs from the purified protein samples, we used antihuman antibody 2A11 (Hytest Oy, Finland) that we received from University of Turku as Europium labeled (Eu-2A11). We first diluted the eluents from previous purification steps in 1:100-1:10000 ratio into Assay Buffer (Kaivogen Oy, Finland), after which 200 µl of a dilution was added per well. A standard Fab with known concentration was diluted also in 0.05 - 1.5 ng/µl Assay Buffer to be able to calculate the Fab concentrations. We usually made three same samples to calculate the average for the signal. The dilutions were then incubated on goat antihuman plates for 30 - 60 min in RT on a plate shaker. Unbound proteins were washed with Delfia plate washer (Wallac Oy, Finland), after washing 50 ng of Eu-2A11 per well in Assay Buffer was added. The plate was then incubated and washed as previously. Finally 200 µl of Enhancement solution per well was added and incubated for 10 min in RT while shaking prior measuring the time-resolved fluorescence of the Europium with Victor or Hidex fluorometry.
To calculate the Fab concentration, we made a standard curve from the standard Fab concentrations and signals. From the linear fit we were able to calculate the Fab concentration in our samples.
We made our standard curve from three concentrations of the standard Fab, because the rest of the higher concentrations saturated the signal already. From the curve below we took the equation f(ng/µl)=(signal-117,870)/1,905, from where you get the concentration by inserting the fluorescence.
Figure 3. The standard curve to determine the Fab concentrations in the samples.
According to our results we got Fabs as can be seen in the figure below.
Figure 4. Amount of Digoxigenin Fabs in our samples.
Comparing site-specific and unspecific immobilization
This was the final and the most important measurement in this project! Beads with site-specifically coated antibodies versus unspecifically coated beads.
Site-specifically and unspecifically coated beads were saturated with Cy5 labeled Digoxigenin (University of Turku). Cy5 signal intensity and variation was measured with flow-cytometer.
Flow cytometer was set to count 100 000 beads and excitate Cy5-label with 650 nm light and measure 670 nm emission. Core-size was set to 10 µm and flow rate 14 µl/min. Results were analyzed with FlowJo.
Random conjugations caused severe cross linking and uneven coating of the magnetic beads which increased the variability of the assay dramatically. This all showed that our method of conjugation is far more superior to conventional random conjugation strategies. Also the simplicity of click chemistry makes it much easier to establish repeatable biosensor preparation and manufacturing protocols for anything spanning from prototypes to industrial scale.
Figure 5. (A) The size (Forward scatter, FSC-area) and shape (Side scatter, SSC-area) distribution of randomly and site-specifically immobilized beads. This shows how a site specifically labeled antibody with only one reactive site does not cross-link the beads in the same manner as a randomly conjugated antibody. (B) A histogram of fluorescence intensity of Cy5 at 700nm. the total population 100000 beads. Uncoated beads in red. Passively adsorbed beads in blue, random conjugation in orange and site specific conjugation using pAzF in green. A much larger variance is achieved when using random conjugation because of the crosslinking of beads. Site specifically coated beads are more uniform. (C) when monomeric beads are measured, a much more uniform coating is once more observed with virtually no unspecific binding or empty beads with a huge difference in variance. Variance of the assay is a critical factor for the sensitivity and repeatability of any test.
Temperature stability measurements of Fab fragments
Melting temperature of Fabs was performed with Prometheus NanoDSF differential scanning fluorometer (Munich, Germany). These measurements were kindly made possible to us by our sponsor PerkinElmer Turku. The proteins were measured as is, in their current buffers (PBS) using a capillary tube to collect 10 µl of the sample for the assay run.
Figure 6. (A) Melting temperatures of Fabs are measured as a ratio of fluorescence at 330nm and 350 nm. When the proteins' tertiary structure starts to unfold, the proportion of 330nm fluorescence sharply increases. (B) This can be also displayed as a derivative of that ratio. Peak of this function shows Tm of proteins. The temperature stability of Fab with a pAzF at the histag does not alter the stability of Fab.
Figure 7. Change of light scattering as a function ot temperature was measured to determine the aggregation of our Fabs. Fab with a pAzF modification shows a drastic tendency to aggregate at higher temperatures. This is caused by the cross-linking nature of pAzF at UV light and high temperatures. This aggregation coincides with the unfolding of tertiary structure of the protein. Normally these aggregation curves do not exhibit this sort of rapid ascension in the curve. We speculate that you could use these sort of measurements also as tool to test pAzF incorporation into proteins.