iGEM 2019 — Team Rice

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Rice/Notebook

[ ]

* Project
  [ ]
  + [Description](/Team%3ARice/Description)
  + [Parts](/Team%3ARice/Parts)
  + [Safety](/Team%3ARice/Safety)
  + [Medal Requirements](/Team%3ARice/Medal)
  + [Judging Form](https://igem.org/2019_Judging_Form?team=Rice)
* Wetlab
  [ ]
  + [Design](/Team%3ARice/Design)
  + [Experiments](/Team%3ARice/Experiments)
  + [Results](/Team%3ARice/Results)
  + [Protocols](/Team%3ARice/Protocols)
  + [Notebook](/Team%3ARice/Notebook)
  + [Contribution](/Team%3ARice/Contribution)
  + [Improve](/Team%3ARice/Improve)
* Drylab
  [ ]
  + [Thermometer Design](/Team%3ARice/Model)
  + [Software](/Team%3ARice/Software)
  + [Thermometer Analysis](/Team%3ARice/DrylabAnalysis)
* Human Practices
  [ ]
  + [Impact Report](/Team%3ARice/ImpactReport)
  + [Integrated HP](/Team%3ARice/Human_Practices)
  + [Meta-Analysis](/Team%3ARice/Meta_Analysis)
  + [Education](/Team%3ARice/Public_Engagement)
  + [Collaborations](/Team%3ARice/Collaborations)
* Team
  [ ]
  + [Members](/Team%3ARice/Team)
  + [Advisors and PIs](/Team%3ARice/Advisors_PIs)
  + [Attributions](/Team%3ARice/Attributions)

#

### [Download a PDF of our lab notebook here!](https://static.igem.org/mediawiki/2019/b/b1/T--Rice--LabNotebook.pdf)

# Wetlab Timeline

## June

**4-6**: Wetlab bootcamp! We all got trained in basic wetlab techniques
**7-31**: Lots of research went into designing our project. We met almost everyday to read papers and design constructs

## July

**9**: Began inoculation of borrowed plasmids
**11**: Acquired the template, which is referred to as pBEST, that we would be using to make a pCON-araC
**12**: Restriction digests have been failing. We realized today it was due to bad enzymes and loading dye. We ordered new supplies
**13**: We performed annealing reactions to make some of our parts, including otsA and otsB, iaaM and iaaH and some of the preliminary thermometers
**14**: Redid the inoculation of the borrowed “part plasmids”
**17**: Attempted our first Golden Gate of our parts plus the borrowed part plasmids. Later results would show this did not work
**22**: Redid the anneals, this time doing phosphorylation reaction first. We decided to make an otsBA operon by PCRing off of an E.coli genome. Attempted another Golden Gate to make a pCON-araC. Later results would show this failed
**23**: Since our constructs need to be arabinose induced to fluoresce green, our plates only had white colonies, we decided to pick a few and digest them to see what they were. We determined that they were all unreacted ampR plasmid. To try to improve our chances of picking the right colonies, we both plated in arabinose and performed a SwaI digest before transforming. The SwaI digest selectively cuts AmpR
**24**: Transformed SwaI restriction digest of pIG102 into DH108 comp cells and plated on LB-amp plates and LB-amp-ara plates but they did not show any growth when checked on later
**25**: Retransformed pIG102 and pIG102 SwaI digest. Conducted Golden Gate assembly of linIG102 and pIG101 to make pIG102, transformed them, and plated them on LB-amp plates. We modified our miniprep protocol because we had been getting such low DNA concentrations by lowering the amount of elution buffer
**26**: We realized our cassettes were wrong after doing a notI restriction digest. We sent our parts in for sequencing to see what was the issue
**28**: Transformation of pIG005 failed
**31**: Prepared P. putida comp cells and plated them on LB plates and two PNS plates with varying levels of sucrose. LB plate showed significant growth and the PNS plates + sucrose showed no growth

## August

## September

## October


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Rice/Experiments

[ ]

* Project
  [ ]
  + [Description](/Team%3ARice/Description)
  + [Parts](/Team%3ARice/Parts)
  + [Safety](/Team%3ARice/Safety)
  + [Medal Requirements](/Team%3ARice/Medal)
  + [Judging Form](https://igem.org/2019_Judging_Form?team=Rice)
* Wetlab
  [ ]
  + [Design](/Team%3ARice/Design)
  + [Experiments](/Team%3ARice/Experiments)
  + [Results](/Team%3ARice/Results)
  + [Protocols](/Team%3ARice/Protocols)
  + [Notebook](/Team%3ARice/Notebook)
  + [Contribution](/Team%3ARice/Contribution)
  + [Improve](/Team%3ARice/Improve)
* Drylab
  [ ]
  + [Thermometer Design](/Team%3ARice/Model)
  + [Software](/Team%3ARice/Software)
  + [Thermometer Analysis](/Team%3ARice/DrylabAnalysis)
* Human Practices
  [ ]
  + [Impact Report](/Team%3ARice/ImpactReport)
  + [Integrated HP](/Team%3ARice/Human_Practices)
  + [Meta-Analysis](/Team%3ARice/Meta_Analysis)
  + [Education](/Team%3ARice/Public_Engagement)
  + [Collaborations](/Team%3ARice/Collaborations)
* Team
  [ ]
  + [Members](/Team%3ARice/Team)
  + [Advisors and PIs](/Team%3ARice/Advisors_PIs)
  + [Attributions](/Team%3ARice/Attributions)

#

[![](https://static.igem.org/mediawiki/2019/d/d4/T--Rice--cloningicon.png)](#Clo)

[![](https://static.igem.org/mediawiki/2019/8/83/T--Rice--RNATiconred.png)](#RNA)

[![](https://static.igem.org/mediawiki/2019/2/22/T--Rice--Bacteriagrowthicon.png)](#BG)

[![](https://static.igem.org/mediawiki/2019/e/ef/T--Rice--planticongreen.png)](#PG)

# Cloning

In order to design our circuits for testing the enzymes and thermometers outlined on the [Project Design page,](https://2019.igem.org/Team%3ARice/Design)we carried out the following process:

1. Obtained the linear DNA templates for the parts and added BsaI and Esp3I overhangs
2. Inserted the part into a vector using Golden Gate assembly to make a part plasmid
3. 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.

![](https://static.igem.org/mediawiki/2019/9/90/T--Rice--spotlabeling.svg)

![](https://static.igem.org/mediawiki/2019/6/63/T--Rice--spot25.svg)

![](https://static.igem.org/mediawiki/2019/2/20/T--Rice--spot30.svg)

## 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.](https://2019.igem.org/Team%3ARice/Results#PG) Testing *A. thaliana* Root GrowthMotivation 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](https://2019.igem.org/Team%3ARice/Results#PG) 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 constructsMotivation 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.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Rice/Results

[ ]

* Project
  [ ]
  + [Description](/Team%3ARice/Description)
  + [Parts](/Team%3ARice/Parts)
  + [Safety](/Team%3ARice/Safety)
  + [Medal Requirements](/Team%3ARice/Medal)
  + [Judging Form](https://igem.org/2019_Judging_Form?team=Rice)
* Wetlab
  [ ]
  + [Design](/Team%3ARice/Design)
  + [Experiments](/Team%3ARice/Experiments)
  + [Results](/Team%3ARice/Results)
  + [Protocols](/Team%3ARice/Protocols)
  + [Notebook](/Team%3ARice/Notebook)
  + [Contribution](/Team%3ARice/Contribution)
  + [Improve](/Team%3ARice/Improve)
* Drylab
  [ ]
  + [Thermometer Design](/Team%3ARice/Model)
  + [Software](/Team%3ARice/Software)
  + [Thermometer Analysis](/Team%3ARice/DrylabAnalysis)
* Human Practices
  [ ]
  + [Impact Report](/Team%3ARice/ImpactReport)
  + [Integrated HP](/Team%3ARice/Human_Practices)
  + [Meta-Analysis](/Team%3ARice/Meta_Analysis)
  + [Education](/Team%3ARice/Public_Engagement)
  + [Collaborations](/Team%3ARice/Collaborations)
* Team
  [ ]
  + [Members](/Team%3ARice/Team)
  + [Advisors and PIs](/Team%3ARice/Advisors_PIs)
  + [Attributions](/Team%3ARice/Attributions)

#

[![](https://static.igem.org/mediawiki/2019/8/83/T--Rice--RNATiconred.png)](#RNA)

[![](https://static.igem.org/mediawiki/2019/e/ef/T--Rice--planticongreen.png)](#PG)

[![](https://static.igem.org/mediawiki/2019/4/4e/T--Rice--Conclusioniconorange.png)](#Con)

# RNA Thermometer Results

To test the existing kit thermometers and our computationally designed thermometers *in vivo*, we made an RNA thermometer testing circuit, transformed it into *E. coli*, and tested the fluorescent output at different temperatures. These graphs show the relative fluorescent intensity of each thermometer between either 25 °C and 30 °C or 25 °C and 37 °C. Only thermometers that performed better than the positive control were included. "K" stands for the kit thermometers, and "NC" stands for our computationally designed NoChill series of thermometers registered as a part. "V" thermometers are thermometers designed and tested but not registered. For additional thermometer testing data, please reference the [contribution page.](https://2019.igem.org/Team%3ARice/Contribution)

As the first graph shows, most thermometers had about a two-fold change in expression between 25 °C and 30 °C. Additionally, most did not perform better than the existing kit thermometer. Since a 5 °C change in temperature is not that much, we also tested the difference between 25 °C and 37 °C, and the second graph shows that most of the thermometers in that experiment had around a four to six–fold expression change.

![](https://static.igem.org/mediawiki/2019/1/11/T--Rice--3025v2.png)

**Figure 1a**: The ratio of Fluorescence per OD between the temperatures of 30°C and 25°C. The horizontal dash line is used to compare the thermometers we made to that of the best performing kit thermometer.

![](https://static.igem.org/mediawiki/2019/f/f7/T--Rice--rltmcherryexpression2530.png)

**Figure 1b**: The ratio of Fluorescence per OD between the temperatures of 30°C and 25°C. The horizontal dash line is used to compare the thermometers we made to that of the best performing kit thermometer.

![](https://static.igem.org/mediawiki/2019/6/6e/T--Rice--3725v2.png)

**Figure 2**: The ratio of Fluorescence per OD between the temperatures of 37°C and 30°C. The horizontal dash line is used to compare the thermometers we made to that of the best performing kit thermometer.

![](https://static.igem.org/mediawiki/2019/f/f5/T--Rice--nochil05.png)

**Figure 3**: The ratio of Fluorescence per OD between the temperatures of 37°,30°C and 25°C.

# Plant Growth Results

## Determining Plant Media Conditions

The lab that helped us with our plant experiments grew *Arabidopsis thaliana* in PNS media. However, we wanted to make sure that our *Pseudomonas putida* would also be able to grow in the media, because it cannot use sucrose as the sole carbon source. We tested the effect of sodium, sodium citrate, glucose, and glucose+sodium on *Pseudomonas* growth. The bacteria grew in all conditions except for just sodium, so for ease, we decided to add glucose to our plant medium.

![](https://static.igem.org/mediawiki/2019/5/5b/T--Rice--Putidagrowth.jpeg)

We needed to make sure that adding glucose would not adversely affect plant growth. To do this we tested the effect of two different glucose concentrations on plant and bacteria growth and found that the plants grew noticebly more at 0.01% glucose.

![](https://static.igem.org/mediawiki/2019/f/f9/T--Rice--arabidopsisputida.jpeg)

# Conclusion

Our results showed that the software we designed is capable of generating usable, and potentially improved, thermometers. While we generated a good deal of useful control data from the plant experiments, the plants did not grow long enough to make any claims about the effectiveness of the enzymes on plant growth.