Goals of the Interlab
Standardization is one of the driving principles of synthetic biology. A rigorous characterization of devices and parts is required for synthetic biology to become a true engineering discipline, in much the same way that electrical engineers rely on a fixed set of standardized parts (transistors, resisters, wires, etc). This includes a standard way of making measurements and expressing quantities in the same way across labs around the world. The Interlab study serves to test a number of devices (six this year) in a multitude of labs, on a variety of plate readers and flow cytometers, in an effort to characterize their function using green fluorescent protein (GFP) as a reporter. The six devices were transformed into bacteria, which were then grown up over a time period of six hours. Fluorescence and absorbance were measured at 0h, 2h, 4h and 6h and compared to the positive and negative controls, as well as a plate control (LB alone). The iGEM InterLab study aims to standardize the way that fluorescent measurements are made, allowing for easier comparisons. All of the tested BioBricks are plasmids (devices were inserted into the standard pSB1C3 backbone with chloramphenicol resistance).
Negative Control - BBa_R0040
Positive Control - BBa_I20270
Test Device 1 (TD1) - BBa_J364000
Test Device 2 (TD2) - BBa_J364001
Test Device 3 (TD3) - BBa_J364002
Test Device 4 (TD4) - BBa_J364003
Test Device 5 (TD5) - BBa_J364004
Test Device 6 (TD6) - BBa_J364005
The InterLab Kit included two controls, a positive and negative control. The Positive Control consists of the constitutively expressed GFP device (I20270) and the Negative Control consists of the pTetR promoter (R0040) with no coding sequence downstream of it (no GFP/reporter).
Materials and Methods
Plate Reader: Promega GloMax Multi+
Absorbance readings were taken at 600nm, while fluorescence readings were taken with the Blue Optical Kit to measure GFP. The Blue Optical Kit has an excitation wavelength of 490nm, and an emission wavelength of 510-570nm. This allowed us to measure GFP production (BBa_E0040), which has an excitation max of 501nm and an emission max of 511nm. This plate reader has an orbital averaging of 1mm, uses a single flash per well, and is a top-optical reader. This plate reader does not use path length correction.
Plate: black with flat, clear bottom
Calibration with LUDOX
To obtain a 'ratiometric conversion factor to transform [our] absorbance data into a standard OD600 measurement', we measured LUDOX HS40. Briefly, Ludox S30 from the InterLab Measurement Kit was thawed. 100μl of LUDOX HS40 was added to our black, flat and clear-bottomed plate, in wells A1, B1, C1 and D1. The same was repeated with dH2O in wells A2, B2, C2 and D2. Using the Promega GloMax Multi+, we took absorbance readings at 600nm, and these data were used to produce a correction factor. The correction factor would later be used to transform the measured OD600 raw data to standard OD600 measurements.
Generating a Fluoroscein Standard Curve
The Fluoroscein Stock from the InterLab Measurement Kit was centrifuged (5 seconds, 5,000 rpm) to pellet the mix to the bottom of the tube. 1ml of 1xPBS was added to the tube to make a 2x fluoroscein solution (100 µM). We then pipetted the solution up and down until the entirety of the 2x fluoroscein solution was dissolved. We next diluted this 2x fluoroscein solution in a 1:1 ratio with 1xPBS to obtain our 1x fluoroscein solution (50 µM). 200μl of the 1x solution was added to wells A1, B1, C1 and D1 in our 96-well plate. 100μl 1x PBS was added to wells A2-12, B2-12, C2-12, D2-12. 100μl from A1 was transferred to A2. The new dilution was mixed and the process was repeated until A11 was reached. 100μl out of the final 200μl in well A11 was discarded. The plate was shaken very gently to ensure that the bottom of all wells were covered with solution and to ensure homogeneity of our fluorescence readings. The process was repeated for rows B, C and D. We then measured the fluorescence of these serial dilutions using the Blue Optical Kit on the Promega GloMax Multi+ plate reader. Data was exported to the Excel Data Sheet from iGEM and the calibration curve was created. Results from this Fluoroscein Standard Curve are below.
210rpm on shaker, dilute to 0.2 then do a 10-fold dilution (once we confirmed that OD600 was linear).
The eight wells in the plate containing the plasmid constructs were taken out of the InterLab Measurement Kit and centrifuged briefly at a low speed to deposit the liquid to the bottom of the tube. 10μl of MilliQ ultrapure water was added to each of the construct wells. The reconstituted plasmid DNA was stored at -4°C.
E. coli (DH5α strain) cells were made chemically competent (Mix & Go E. Coli Transformation Kit and Buffer Set) with a transformation efficiency of 10^5. Aliquots of 100μl were taken out of the -80°C freezer and thawed on ice (~-4°C). Plasmid DNA and chloramphenicol (stock solution of 30mg/ml) were put on ice as well for thawing. Once the 100μl of DH5α E. coli had thawed, 5μl of plasmid DNA was added. The mixture was left on ice for 30 minutes.
LB agar plate preparation
While the competent bacteria and DNA mixture was on ice, Lysogeny Broth (LB) plates were made. Low-salt LB agar was autoclaved for 20 minutes. Once the agar was completely liquefied, this liquid LB was placed in a 68°C water bath in order to cool down. After reaching the target temperature, the bottle containing the broth was allowed to cool below 68°C for 3-5 minutes in order to add chloramphenicol. Chloramphenicol was then added in a 1:1000 ratio to the LB (1μl of chloramphenicol per ml of LB). In a flow cupboard, the solution was poured into Sterile Standard single-use petri dishes (25ml per dish) and allowed to resolidify overnight. After 30 minutes on ice, the bacterial/plasmid DNA solutions were heat shocked in a 42°C heat block for 45 seconds. They were put on ice again for 1 minute and then 100μl of the iGEM supplied SOC was added. This was then incubated at 37°C for 1 hour at 400rpm. After the end of incubation, 100μl of the solution for each part was added on separate LB agar plates to make the plates. The solution in each plate was spread using sterile glass beads. All the plates were taped and placed in a 37°C incubator overnight.
The following day, plates were taken out of the incubator and stored in a refrigerator unit set to 4°C. In the afternoon, two colonies for each constructed were picked from their respective plates, allowed to grow up for about 16h in LB+chloramphenicol, and then placed in long-term storage at -80C. 10ml of liquid LB+chloramphenicol was pipetted into sterile glass test tubes, two test tubes for each construct (16 in total). Using P200 pipette tips, 2 colonies for each construct were picked from their respective plates. The test tubes were placed on a large, spinning wheel set to 210rpm in a 37°C incubator for 15 hours.
Extracting plasmid DNA
A QIAprep Spin Miniprep Kit (250) was used to extract the plasmid DNA from the overnight cultures. The concentration of the extracted DNA solution was measured using Thermo Scientific’s NanoDrop 1000 spectrophotometer by blanking it first with elution buffer and then adding 2μl of plasmid solution, to ensure that we successfully extracted some sort of nucleic acid.
Cell Measurement Protocol
Initially, the OD600 of the overnight cultures was measured on a Thermo Scientific NanoDrop 1000 machine, set to the ‘Cell Culture’ reading. We then diluted all of the overnight cultures (16 in total) to a concentration of 0.2, using our linear curve calculations. We found that diluting the overnight cultures directly to 0.02 resulted in unreliable readings on the NanoDrop. After diluting each culture to 0.2, we diluted each 10-fold, and immediately withdrew 500ul from each of the 0.02-concentration overnight cultures to use as our 0 hour readings. The rest was immediately placed in a 37C incubator with a horizontal plate movement speed of 210rpm. Aliquots were removed and loaded onto the plate at 2 hours, 4 hours, and 6 hours. The plate loading, which applies for every time point, is below. Both absorbance (600nm) and fluorescence (Blue Optical Kit, 490 excitation, 510-570 emission) measurements were taken on the plates. The same plate was washed thoroughly after every use, and re-used for each of the time points, as it had previously been thoroughly characterized and calibrated, and we wanted to ensure that the plate did not give inconsistent or unreliable readings between time points. The data was recorded in the iGEM Data Excel Sheet and sent to email@example.com.
Results and Discussion - All Wrapped Into One!
At each time point, we took a 'raw absorbance' reading on the Promega GloMax Multi+ machine. The figure below shows the results from these 600nm absorbance readings for each device (two replicates per device). The OD600 measurements seem to be typical for all devices; namely, all of the bacterial cultures grew up at a somewhat even pace. Of course, depending on the burden of each device, the growth rate of the corresponding transformed bacterial culture could be slowed. Since all of the devices were of approximately equal length (~900 bp), we would not expect to see significantly different growth rates. As expected, we observed no increase in the ABS600 of the LB+chloramphenicol plate controls. Device 4 replicates did have less growth than the other bacterial cultures, though the reason for this is unclear.
Next, we measured the fluorescence 'output' for each of the devices, again in two replicates for each of the devices, over each of the time points. These data, unlike the absorbance 600nm values, do suggest marked differences between the different devices. Perhaps most notably, Device 3 and Device 6 had negligible increases in fluorescence over time. After closely investigating each of the six experimental devices, we found that Device 3 (BBa_J364002) and Device 6 (BBa_J364005) have the same promoter, part J23117. This promoter is not shared by any of the other devices. When this promoter was investigated more closely, we found that it was designed by the 2006 UC-Berkeley team and is a 1bp mutant of J23114, a promoter which is already a very weak promoter. Furthermore, we found a prior characterization of this exact promoter, as it was used to characterize RFP fluorescence in a previous year (see http://parts.igem.org/Part:BBa_J23114). The J23117 promoter has been characterized with a value of 162 (a.u.) while some of the other promoters used in this Interlab study are as high as 1800+
After exporting all of our plate reader measurements into the provided Interlab Excel spreadsheet, we calculated the fluorescence minus the background, at each time point, for each of the replicates and devices. Given our observations in the fluorescence readings, including the observation that Device 3 and Device 6 did not have increased fluorescence over time, it was really no surprise that their fluorescence minus background measurements were close to zero, or negative, at each of the time points. The devices with the highest 'normalized' fluorescence output were the positive control (of course), Device 1, and Device 2. These normalized data are provided in the figure below.
Suggestions for Interlab Improvements and Challenges Encountered
Suggestions for Improvements
As we went through the protocol, there were a few things that we encountered that we felt could have been improved. Most of the following recommendations relate to the actual written protocol.
1) Greater clarity in details. Instead of writing "Do the same steps for columns B, C and D", provide a detailed schematic which shows every single well and the total volume of solutions in that well. Also, it should be written in the protocol to vortex the bacterial cultures before taking aliquots at each time point. We neglected to do this in one of our early trials, and we had readings come out 'all over the place' because our bacterial cultures settled a bit at the bottom despite the shaking.
2) Access to comparison data. For example, when we were performing the initial LUDOX measurements, we felt that our read-outs were extremely low values. By providing access to some examples of past data, we could more quickly troubleshoot problems such as these.
3) Further standardization of plates. Would it be possible, in the future, to ship plates along with the kits so that all teams have relatively equal optic readings? We tried a couple of different plates, and some worked better than others! We obtained our best results with black, flat and clear-bottomed plates.
1) It was, at times, difficult for us to figure out the standard settings on our plate reader. Despite consulting the manual, we ended up calling Promega three different times to find information such as the orbital averaging, number of flashes per well, etc.
2) We repeated the Interlab four times (yes, four times!) because, on each occasion, we made one small error. In early trials, we either had uneven pipette loading, failed to vortex colonies before aliquoting, or did not shake our bacterial cultures fast enough (which led to quite low Abs600 readings at the later time points).
3) We found that it was best to have a small group of students assigned to the Interlab. This way, each person is consistent in their given task. A single individual loaded the plates at every time point, while another individual vortexed and aliquoted, and yet another individual handled the plate reader. By keeping these tasks consistent, we avoided the possibility of different people performing an identical task in different manners!
All images on this page were made with GraphPad PRISM 7.0 software.
Team members that contributed to the Interlab: See Attributions page