Team:Rice/Experiments
Cloning
In order to design our circuits for testing the enzymes and thermometers outlined on the Project Design page,we carried out the following process:
- Obtained the linear DNA templates for the parts and added BsaI and Esp3I overhangs
- Inserted the part into a vector using Golden Gate assembly to make a part plasmid
- Performed multi-part Golden Gate assemblies to make gene cassettes of the circuits
Linear DNA synthesis
Linear DNA synthesis of the plant growth promoting enzymes
We obtained IaaMH by taking the DNA from the BBa_K515100 biobrick found in the kit. In order to add the correct overhangs, we did 3 separate PCRs using primers oIG005/ oIG006, oIG007/oIG008, oIG009/oIG010. AcdS was obtained by ordering a gene block on IDT. OtsBA was obtained by amplifying DNA off of the E. coli genome using oIG042/oIG043. Sequences for these primers and parts can be found on the parts page.
Linear DNA synthesis of the inducer
Motivation and Objective
After doing preliminary research, we decided that the best inducer to use was araC and arabinose, since P. putida can not catabolize arabinose. We also decided to use the origin RK2 since it was well documented to perform well in P. putida.
Methods
One of our advisors gave us a plasmid called pBEST that contained Pcon-araC. We designed primers to amplify Pcon-araC from this plasmid and purified the PCR product.
Results and Conclusion
After several gel purification failures, we decided to look at the linear DNA sequence and its primers, and we discovered that the primers bind nonspecifically to other terminators in the template. We continued to troubleshoot for several days with no luck, so we ordered a gBlock of Pcon-araC.
DNA Assembly
Motivation and Objective
Through Golden Gate assembly, we attempted to ligate araC into the vector pSPB440 with AmpR, RK2 origin, and a sfgfp dropout. After trying to get colonies from a vector containing araC and RK2 and failing for two months, we ran an experiment with positive controls.
Methods
We built a series of 4 vectors containing a sfgfp dropout, ampicillin resistance, either araC in the vector or a double terminator, which served as the control, and either RK2 or p15A as the origin.
Results and Conclusion
The results indicated that only those with the p15A origin produced the correct colonies. This told us that the problem was not with our araC but instead was with the RK2 origin we were using. Therefore, we switched to another broad host origin.
RNA Thermometer Characterization
Testing rationally designed thermometers in time course
Motivation and Objective
In order to get familiar with the protocol of testing thermometers, we tested the thermometers that were made using a ‘rationally designed’ process. The rationally designed thermometers were created by manually adding and removing base pairs in the complementary sequence to the RBS to lower the melting points.
Method
We inoculated 2mL of pIG015, 016, 018, 019, 029 in LB30 enriched with 1mM of MgSO4. These were left to grow for 8 hours at 37°C in a 96 well block. We measured the OD600 of the saturated culture in a 1:30 dilution and then diluted the saturated culture to a final OD600 of 0.01. These were then grown for approximately 2 hours until their OD600 hit 0.1. In the meantime, Corning 3904 black clear flat bottom 96 well plate were prepped by added 100µL of 2 mM IPTG LB. 100µL of each culture was added to the appropriate well. The plate was then grown in the plate reader at 30°C for 6 hours. This entire process was repeated the next day, but the plate was grown at 25°C for 6 hours instead.
Results and Conclusion
While this experiment helped us refine our testing procedure, the results were not guaranteed for each thermometer due to significant cross contamination between wells. Factoring this in, it was still clear that almost none of these thermometers showed a substantial improvement compared to the controls. We decided to abandon these ‘rationally designed’ thermometers for the computationally designed thermometers.
Testing in solid culture using spot inoculation
Motivation and Objective
We decided to do a solid culture test of the first round of thermometers in order to get a visual representation of the fluorescence changes across temperatures.
Method
1 µL of saturated culture from each thermometer was spotted onto a LB Kan plate. Images were taken using a macroscope. The thermometers that eventually showed to be promising have been renamed. All others retain the old naming conventions.
Results and Conclusion
The images turned out to be difficult to quantify, but general observations could be made. For example, RNAT022 and RNAT023 showed to be especially leaky.
Measuring only the end time point: Round 1
Motivation and Objective
Each time the software that predicts the thermometers is ran it outputs a different sequence. We decided to test roughly 25 of the thermometers. Since we had over 20 different constructs to test, it didn’t make sense for us to do a time course for each one. The limitation for this came down to the fact that we only have access to one plate reader. We had more thermometers than could fit into any one plate reader. This coupled with the fact that each temperature would need its own day in the plate reader would mean that it would take us about a week of running experiments before we got data. By only measuring the end point of time, we could get a general idea of the performance of each thermometer in a single day and then retest the best performing thermometers afterwards.
Method
We inoculated 2mL of LB Kan enriched with 1mM MgSO4 with each of the thermometer cassettes and controls. We grew each construct to saturation. We then added 3µL of each saturated culture to 300µL of LB Kan with 1mM IPTG and 1mM MgSO4 in 96 well blocks and let this grow overnight. In the morning we analyzed the data using Spark on the plate reader.
Results and Conclusion
What was then named RNAT002,6,7,18 and 21 all showed improved Fluorescence/OD600 change from 25°C and 30°C in comparison to the Kit thermometers. RNAT020 showed improvement from 30°C to 37°C. The graphs and other information from the experiment can be found on the results page.
Measuring using ramping temperatures
Motivation and Objective
We wanted to use a ramping temperature protocol to more closely mimic a natural environment.
Method
We grew the best performing thermometers, those that have now adopted the no chill naming conventions, till saturation and then diluted to an OD600 of 0.01. After they grew back to an OD600 of 0.1, we added 100µL of culture to 100µL of 2mM IPTG LB Kan in a 96 well plate. They were then placed in the plate reader. The plate reader spent ~5 hours at 25°C, ~4 hours at 30°C and then ~ 2 hours at 37°C.
Results and Conclusion
The plate reader failed to record the fluorescence for any of the thermometers. While we did not have time to repeat this experiment, we strongly recommend any other iGEM team seeking to improve or characterize RNA thermometers to attempt a similar experiment.
Measuring only the end time course: Round 2
Motivation and Objective
In order to get additional replicates from the first round of testing, we decided to redo the end time course experiments
Method
We inoculated 900µL of LB kan enriched with 1mM MgSO4 from glycerol stocks of the best performing thermometers. IPTG was added to ¾ of all replicates. They were grown for 10 hrs and then analyzed via a plate reader.
Results and Conclusion
P. putida Experiments
Testing P. putida F1 MIC for Carbenicillin and Ampicilin
Motivation and Objective
It was important for us to use as little antibiotic as possible, since Carbenicillin, a β inhibitor, has relatively mild effects of plant growth still affects plant growth. We set out to test how much antibiotic we could use while still maintaining selectivity of P. putida .
Methods
We grew both P. putida transformed with a plasmid containing AmpR and wildtype in various concentrations of Carbeniciliin and Ampicillin ranging from 0.5 mg/L to 50 mg/L.
Results and Conclusions
Wild type P. putida grew in concentration of 2.5 mg/L and under of Ampicillin and Carbenicilin. We decided to use 5 mg/L of Ampicillin for cloning. This concentration is too high for plant experiments. Thus we did not use antibiotics in the plant experiments, but instead worked in a biosafety cabinet under sterile conditions.
A. thaliana Plant Experiments
Testing A. thaliana Growth with Untransformed P. putida and 0.6% PN Glucose
Motivation and Objective
After deciding to use PN and glucose as our growth media, we needed to make sure that adding glucose would not adversely affect plant growth. To do this we wanted to test the effect of two different glucose concentrations on plant and bacteria growth.
Methods
We made 0.6% PN plates with two different glucose concentrations, 0.01% and 0.1%, plated A. thaliana seeds inoculated with untransformed P. putida and let them grow for a few days and qualitatively observe how these different conditions would effect plant and bacteria growth.
Results and Conclusion
\We found that the plants grew noticebly more at 0.01% glucose, seen on our Results page.
Testing A. thaliana Root Growth
Motivation and Objective
Motivation and Objective
Before we test whether our enzyme cassettes aid plant growth, it was important to make plant growth control experiments in order to have a baseline of plant growth without plant growth promoting bacteria.
Methods
We designed five control experiments with 1% PNS plates:
1. No additives, only 1% PNS
2. Arabinose
3. IAA (auxin)
4. Trehalose
5. Arabinose, IAA, trehalose
Results and Conclusion
Preliminary scans of the plates show sporadic root growth so we deemed the results unusable and we are in the process of repeating these experiments.
Testing A. thaliana with induced and uninduced P. putida containing the enzyme constructs
Motivation and Objective
Motivation and Objective
Once our enzyme constructs were made, they were ready to transform into P. putida and then test to see their effects on plant growth. It was important to do each enzyme construct separately at first to have a baseline of plant growth before testing all enzyme constructs at once. We also decided to have some controls, with all constructs induced with arabinose and IPTG and all constructs without inducers for plant growth comparison with and without induction.
Methods
We inoculated wild type P. putida and P. putida with the separate enzyme constructs (iaaMH (pIG104), acdS (pIG105) and otsBA (pIG106)). Then, we split each construct and the wild type so that half of each construct and the wildtype would be induced with 10 mM arabinose and IPTG and the other half was not. Here is the outline of the experiments we conducted:
1. With no bacteria
2. With P. putida
3. pIG104, induced
4. pIG104, non-induced
5. pIG105, induced
6. pIG105, non-induced
7. pIG106, induced
8. pIG106, non-induced
9. pIG104, pIG105, pIG106, induced
10. pIG104, pIG105, pIG106, non-induced
Results and Conclusion
All plates showed growth, but they were only grown for 6 days, so we cannot draw any valid conclusions based on qualitative observations, so we will continue to let them grow.