iGEM 2021 — Team RUM-UPRM

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# Team:RUM-UPRM/Notebook

RUM-UPRM Wiki Source Code

[RUM-UPRM](https://2021.igem.org/Team%3ARUM-UPRM)![](https://static.igem.org/mediawiki/2021/0/0c/T--RUM-UPRM--minilogordetox2021modificado.png)

**Notebook**

* [Overview](#section1)
* [Cloning Plans](#section2)
* [Laboratory Notebook](#section3)

# Overview

![](https://static.igem.org/mediawiki/2021/thumb/c/cb/T--RUM-UPRM--LAB1.JPG/800px-T--RUM-UPRM--LAB1.JPG.png)

We started working in our designated laboratory during early Spring 2021. Our advisors and PI's were in charge of leading several training workshops to familiarize us with basic laboratory techniques such as safety precautions, culture media preparation, DNA extraction, and gel electrophoresis. Our training lasted around six weeks. Once finished, we began working in the laboratory while simultaneously polishing our cloning plans so that we would have a concrete workflow to follow during summer. During June and July we held several bacterial transformation protocols, and during August 2021 we finally could start following our 3A Cloning workflows. Unfortunately, came across unexpected results and were forced to [change our plans](https://2021.igem.org/Team%3ARUM-UPRM/Engineering#section4), which led us to prepare one final and improved cloning plan using pUC19 as our plasmid. Below you can find all three cloning plans used during this iGEM 2021 cycle.

![](https://static.igem.org/mediawiki/2021/thumb/1/15/T--RUM-UPRM--LAB2.JPG/800px-T--RUM-UPRM--LAB2.JPG.png)

# Cloning Plans

[3A Cloning Device 1](https://static.igem.org/mediawiki/2021/c/cb/T--RUM-UPRM--DEVICE1-3A.PDF)

[3A Cloning Device 2](https://static.igem.org/mediawiki/2021/a/af/T--RUM-UPRM--DEVICE2-3A.PDF)

[pUC19 Cloning Device 1 & 2](https://static.igem.org/mediawiki/2021/a/a7/T--RUM-UPRM--pUC19CloningStrategyD1D2.pdf)

# Laboratory Notebook

We used Benchling to maintain our digital laboratory notebook, which enabled us to keep all of our results and materials organized in the same place. As we worked in the laboratory, we divided our laboratory notebook by weeks (seven day periods, from Sunday to Saturday), but for the purpose of further organizing our work we separated them by month. We kept this format until we started working individually on each device using 3A Cloning. We divided our [Biology Team](https://2021.igem.org/Team%3ARUM-UPRM/Members) members into two different groups so that we could work on both devices at the same time. Their respective laboratory notebooks are classified as **3A Device 1** and **3A Device 2**. We kept this workflow from late August 2021 to the first week of October 2021. Afterwards, we switched over to the pUC19 workflow, and merged both groups. The final laboratory notebook is classified as pUC19 and was worked on during **October 2021**.

[April 2021](https://static.igem.org/mediawiki/2021/5/5e/T--RUM-UPRM--April2021.pdf)

[May 2021](https://static.igem.org/mediawiki/2021/1/13/T--RUM-UPRM--May2021.pdf)

[June 2021](https://static.igem.org/mediawiki/2021/6/69/T--RUM-UPRM--June2021.pdf)

[July 2021](https://static.igem.org/mediawiki/2021/1/1a/T--RUM-UPRM--July2021.pdf)

[August 2021](https://static.igem.org/mediawiki/2021/5/5b/T--RUM-UPRM--August2021.pdf)

[3A Device 1](https://static.igem.org/mediawiki/2021/a/a3/T--RUM-UPRM--Device13ACloning.pdf)

[3A Device 2](https://static.igem.org/mediawiki/2021/b/b6/T--RUM-UPRM--Device23ACloning.pdf)

[pUC19 Device 1 & 2](/)

![](https://static.igem.org/mediawiki/2021/thumb/c/ca/T--RUM-UPRM--LAB3.JPG/800px-T--RUM-UPRM--LAB3.JPG.png)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:RUM-UPRM/Engineering

RUM-UPRM Wiki Source Code

[RUM-UPRM](https://2021.igem.org/Team%3ARUM-UPRM)![](https://static.igem.org/mediawiki/2021/0/0c/T--RUM-UPRM--minilogordetox2021modificado.png)

**Engineering Success**

* [Research](#section1)
* [Design](#section2)
* [Build](#section3)
* [Test](#section4)
* [Learn](#section5)
* [References](#section6)

# 1. Research

Our aim with this project is to find a modern solution to a problem that has been plaguing the citizens of Vieques for several decades. As we all know, the small puertorrican municipality has been dealing with contamination issues due to the bomb detonations that occurred near its several bodies of water, one of these being the Anones Lagoon in Vieques. Several areas of the municipality were used for military training, and were recently declared as a Superfund site (Sanderson et al., 2017).

# 2. Design

Out of all the contaminants present in Vieques, we chose hexahydro-1,3,5-trinitro-s-triazine (RDX) to be the focus of our project. Rosen et al. (2017) held a study to analyze the concentration of RDX and other explosives in the waters in the municipality. Chakraborty et al. 2008 also spoke about how the leaching of explosive compounds, such as RDX, occurs due to unexploded munitions present in the area.

The XplA/XplB gene cluster present in our genetic circuit was first found in Rhodococcus sp., a bacterium with the potential of fulfilling the biodegradation of RDX in aerobic environments (Bernstein et al., 2011). We believe that these two genes will enable us to conceptualize and eventually construct in vitro a bacterial prototype capable of fulfilling this biological degradation of RDX, to serve as a possible solution to the contamination problems found in Vieques.

# 3. Build

Several publications were used as a reference for the analysis and characterization of the XplA/XplB gene cluster (Jackson et al., 2007; Halasz et al., 2012; Cary et al., 2021). Once we obtained the genetic sequence, we started crafting our genetic circuit using SnapGene and Benchling to facilitate the visualization of our genetic devices. This helped us visualize our cloning workflow and choose the restrictions sites to permit the synthesis of our genetic constructs.

![](https://static.igem.org/mediawiki/2021/6/64/T--RUM-UPRM--D1Diagram.png)

**Figure 1.** Cloning workflow for the assembly of Device 1 of our RDX Biodegradation Genetic Circuit, built using SnapGene.

We made sure to build our [genetic devices](%E2%80%9Dhttps%3A//2021.igem.org/Team%3ARUM-UPRM/Design%22) in such a way that would enable us to easily detect their functionality, while simultaneously keeping them under control and only functioning when specific conditions are met. Reporter genes, fluorescent proteins in our case, would aid us in detecting them as our cloned bacteria grows in culture media. Our chosen promoters also ensure that our devices only work in the presence of RDX and their assigned compounds, meaning our genetic circuit won't be able to jumpstart itself unless it is found in the desired environmental conditions. Lastly, our third device contains a killswitch which will serve as a biocontrol to prevent unnecessary cellular proliferation in the environment, enabling cell lysis once our cloned bacteria fulfills its function.

![](https://static.igem.org/mediawiki/2021/c/c3/T--RUM-UPRM--device1.gif)

# 4. Test

Our first attempts to clone our DNA fragments were made using the [Linearized Plasmid Backbones](%E2%80%9Dhttp%3A//parts.igem.org/Help%3AProtocols/Linearized_Plasmid_Backbones%E2%80%9D) sent out in the 2019 iGEM Distribution kits. The three different plasmids present in the kit allowed us to prepare a 3A cloning workflow which would allow us to prepare stocks of our two fragments in two different plasmids and a single plasmid with both of the needed fragments inside of it. Several weeks later we decided on taking a different approach to our cloning techniques and switched our plasmid to pUC19. Each fragment was cloned individually on its own pUC19 plasmid. Lastly, we expect to validate our devices using fluorometric assays in a 96-well plate reader. More information regarding our proof of concept can be found [here](https://2021.igem.org/Team%3ARUM-UPRM/Proof_Of_Concept).

# 5. Learn

Our original [cloning plans](https://2021.igem.org/Team%3ARUM-UPRM/Notebook#section2) could not be fulfilled due to several unforeseen circumstances that appeared while attempting to transform our bacteria to express our plasmids containing our desired DNA fragments. Pink, fluorescent colonies were observed, even on bacterial colonies belonging to plates that didn't contain the pink fluorescent protein gene. We held additional contamination assays to see what exactly happened and why we were obtaining these unexpected results. Our final decision was to change the plasmids we were using, which is why we resorted to working with pUC19 instead to fulfill our cloning strategy.

# 6. References

Cary, T. J., Rylott, E. L., Zhang, L., Routsong, R. M., Palazzo, A. J., Strand, S. E., & Bruce, N. C. (2021). Field trial demonstrating phytoremediation of the military explosive RDX by XplA/XplB-expressing switchgrass. *Nature Biotechnology*, 1-4.

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:RUM-UPRM/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:RUM-UPRM/Results

RUM-UPRM Wiki Source Code

[RUM-UPRM](https://2021.igem.org/Team%3ARUM-UPRM)![](https://static.igem.org/mediawiki/2021/0/0c/T--RUM-UPRM--minilogordetox2021modificado.png)

**Results**

* [Overview](#section1)
* [Device 1 - 3A Cloning](#section2)
* [Device 2 - 3A Cloning](#section3)
* [Pink Colonies](#section4)
* [Cloning using pUC19](#section5)

# Overview

Over the course of August to October, we implemented various cloning strategies to insert our proposed genetic constructs into *Escherichia coli*. Some setbacks in the 3A Cloning caused our team to reconsider other cloning strategies but we decided to select the simple cloning strategy as the most appropriate option to insert our proposed parts. For the last month, we obtained promising results for a possible successful cloning of Device 1, based on the results of an electrophoresis run of pUC19 samples derived from blue colonies of bacteria after a blue/white screening session. These results prompted us to carry out the [Proof of Concept](https://2021.igem.org/Team%3ARUM-UPRM/Proof_Of_Concept) for Device 1 to validate if what we had was truly what we desired.

We prepared a serial dilution of a saturated RDX solution. We added these dilutions to inoculated cultures of LB medium, with possibly cloned Device 1, in the exponential phase of *Escherichia coli*. When RDX was added, and after an overnight incubation, we took absorbance readings and fluorescence with a transilluminator. Afterwards, we performed plasmid extraction, digested it with PstI and ran a gel electrophoresis. Unfortunately, we did not see any fluorescence and the gel did not show our expected results. For future works we will digest the remaining extraction and run another gel electrophoresis to validate if our Device 1 was successfully cloned.

## Device 1 using 3A Cloning

The 3A Cloning strategy for Device 1 started out as a double digestion using the enzymes PstI and EcoRI for both the fragments and the plasmids. However, the r2.1 buffer used only assured a 100% activity for the enzyme of EcoRI, while the enzyme of PstI only had a 75% of activity. This was followed by a ligation protocol to join Device 1 Fragment 1 (D1F1) with the plasmid pSB1A3, and Device 1 Fragment 2 (D1F2) with the plasmid pSB1K3. No growth was observed on any of the plates after the first transformation test run.

For the second attempt, the buffer for the double digestion was changed to r3.1 which had a 100% activity for the enzyme of PstI, while for the enzyme of EcoRI only had a 50% of activity. This new transformation had bacterial growth on two plates that had the enzyme ligase added, while the control plates, which didn’t have ligase, presented no colony growth. Those plates that demonstrated bacterial growth and were possible cloned bacteria were later inoculated, extracted and digested with restriction enzymes to visualize them under a gel electrophoresis. The gel run didn’t demonstrate the gel bands for the fragments indicating that the plasmids recirculated without the fragments and no insert was found. The following transformations started to present pink colony growth and showed unexpected results which were catalogued in the [Pink Colonies section](https://2021.igem.org/Team%3ARUM-UPRM/Results#section4) down below.

## Device 2 using 3A Cloning

The first attempt of 3A Cloning for Device 2 consisted of a double digestion of the two DNA fragments belonging to Device 2. The enzymes PstI and EcoRI were used to digest each plasmid and fragment. Device 2 Fragment 1 was ligated with the plasmid pSB1A3, and Device 2 Fragment 2 was ligated with the plasmid pSB1K3. The r2.1 buffer ensured that EcoRI would have 100% activity while suspended, while PstI only had 75% activity using the same buffer. This meant our results would be at a disadvantage since both enzymes wouldn’t have maximum efficiency percentages. No gel electrophoresis was performed after the digestion, meaning we wouldn’t be able to determine if the plasmids were linearized or still circularized at the moment of attempting ligation.

During the bacterial transformation, growth was observed on all three plates. This suggests that the linearized plasmids might have been circularized since the plate that didn’t contain DNA ligase still had colonies in it. The largest number of colonies were observed in the plate that only contained the vector and DNA ligase added into the ligation tube. Growth was also observed in the plate that contained DNA ligase, the vector and the insert but after realizing a Miniprep reaction with the colonies, no insert was observed in the gel electrophoresis.

![](https://static.igem.org/mediawiki/2021/b/b9/T--RUM-UPRM--device2-1.JPG)

**Figure 1.** From left to right, Control 1 (Only Vector added), Control 2 (Vector and DNA Ligase added), Experimental (Vector, DNA Ligase and Insert added).

Due to uncertainties about the linearized plasmids possibly being circularized, we ran a gel electrophoresis using the stock of the plasmids. The gel electrophoresis showed multiple bands under each well, leading us to conclude that we are dealing with circularized plasmids that are in three different states: normal, supercoiled, and relaxed. We notified the Device 1 team members and our PI’s about this and started optimizing our work plan according to these new findings.

![](https://static.igem.org/mediawiki/2021/4/4a/T--RUM-UPRM--device2-2.JPG)

**Figure 2.** From left to right: Well 1 - 1kb Plus Ladder; Well 2 - pSB1A3, Well 3 - pSB1C3, Well 4 - pSB1K3.

Contrary to our original cloning plan, we decided to switch up the enzymes used in our digestions and switched over to using SpeI and EcoRI, both of which have 100% efficiency in the r2.1 buffer. We also switched over to using the thermocycler for our ligation and transformation reactions to guarantee a more optimal temperature distribution during the reactions and the heat inactivation.

Unfortunately this time we didn’t observe any growth in all of our transformation plates. We left them incubating for over 48 hours and still no colonies were to be found. One last additional transformation was realized and we obtained the same results: zero colonies were observed.

We consulted our results once again with our PI’s and Advisors and, as mentioned above, the conclusion we came to was that the restriction sites on the edges of our DNA fragments didn’t leave enough space for our restriction enzymes to cut efficiently.

### Adapting to situations

##### The transition of cloning strategy from 3A to simple cloning

Throughout our 3A Cloning plan in Device 1, we started noticing irregularities in colony growth after bacterial transformation. For our protocols, we were using the vector pSB1A3 for our Fragment #1 portions, and psB1K3 for Fragment #2 portions. The appearance of pink colonies in our transformation plates caused the team to reconsider our cloning strategy as we did not expect these results. These pink colonies suggest that the cells could be transformed only with mCherry (a red fluorescent protein) or that our plates were contaminated with *Serratia marcescens*, a Gram-negative bacteria known for its red colored colonies.

![](https://static.igem.org/mediawiki/2021/7/75/T--RUM-UPRM--pink.png)

**Figure 3.** Pink colonies observed on all of our pSB1A3 and pSB1K3 plates.

![](https://static.igem.org/mediawiki/2021/d/d0/T--RUM-UPRM--pink1.png)

**Figure 4.** Pink fluorescence observed when placing the plates with pink colonies under UV light using a transilluminator.

Upon closer inspection of our sequences, and with the help of Dr. Carlos Rios Velazquez, and our advisors Victor and Luis Montalvo, we noticed that there were not enough nucleotides for the restriction enzymes to bind onto the fragments of our devices. This would cause digestion to occur with much less efficiency than anticipated. A [new cloning plan](https://2021.igem.org/Team%3ARUM-UPRM/Notebook) had to be developed because of the uncertainties regarding plasmid structure and the color of the colonies. We resorted to simple cloning in which the fragments would be inserted into a plasmid (pUC19) and then digested to create new restriction enzyme cut sites in a way that each fragment would join in a sequential manner during ligation. While carrying out the blue white screening technique during this new plan, we saw pink colonies again, but after several transformations we saw blue and white colonies.

## Device 1 and Device 2 using pUC19 Cloning

**### ➜Device 1**

Device 1 showed promising results in the October electrophoresis. According to the last electrophoresis performed, we decided to carry out a simplified [Proof of Concept](https://2021.igem.org/Team%3ARUM-UPRM/Proof_Of_Concept) using RDX thanks to the doctoral student Francheska Carrero and Dr. Samuel Hernández as the gel showed possible successful cloning of both Fragments of Device 1.

![](https://static.igem.org/mediawiki/2021/a/a8/T--RUM-UPRM--slide9gel.jpg)

**Figure 5.** Gel electrophoresis ran showing the possible cloned plasmids with the Device 1 Fragments.

Each well contains different samples of pUC19 with possible Device 1 digested with PstI. Results shown in this gel indicate unexpected results from the majority of samples except the sample F1F2 2B. This sample showed an intense band around 2.7 kb, which might be pUC19 without the device, another band around 3.1kb which indicates possible successful Fragment cloning of Fragment 1 (1.5kb) and 2 (1.6kb)

An electrophoresis of digested plasmid with possible insertion of Device 1 was carried out with enzymes PstI and XbaI. The expected location of bands are 1.5 kb y 1.6 kb, but we obtained a band around 5.7kb and around 2.7kb. This might indicate that digestion was not done successfully. However, all of the bands above the ones in 2.7 kb are located at around 5.7-5.8 kb, which might represent pUC19 with Device 1.

![](https://static.igem.org/mediawiki/2021/1/19/T--RUM-UPRM--Slide8gel.jpg)

**Figure 6.** Gel electrophoresis ran showing the possible pUC19 plasmids with the Device 1 Fragments.

For our [Proof of Concept](https://2021.igem.org/Team%3ARUM-UPRM/Proof_Of_Concept) we prepared a serial dilution of a saturated solution of RDX in 70% ethanol, and added this to our *Escherichia coli* in its exponential phase. We saw an inverse relationship between the concentration of RDX in solution and bacterial growth, also seen in our absorbance testing. Also we observed our cultures for fluorescence with a transilluminator after an overnight incubation.

After the observation for fluorescence and absorbance readings, we decided to perform extraction, followed by a digestion with enzyme PstI and a gel electrophoresis protocol. We did not see the expected fluorescence on any of the incubated bacteria, so we determined that mCherry was perhaps not being expressed. Additionally, the gel electrophoresis did not show the expected results. In the future, we will perform another digestion with the enzyme PstI and a gel electrophoresis protocol to answer the following questions: *Were there mistakes with the gel electrophoresis protocol? Was Device 1 successfully cloned into pUC19?*
![](https://static.igem.org/mediawiki/2021/e/e6/T--RUM-UPRM--Cell_Growth_in_RDX.JPG)

**Figure 7.** Device 1 *Escherichia coli* DH5alpha Cell Growth in RDX Solution.

![](https://static.igem.org/mediawiki/2021/c/cd/T--RUM-UPRM--BacGrowthSamplesC.jpeg)

**Figure 8.** Bacterial Growth of *Escherichia coli* with RDX Samples 1C-6C (left to right).

![](https://static.igem.org/mediawiki/2021/8/89/T--RUM-UPRM--BacGrowthSamplesE.jpeg)

**Figure 9.** Bacterial Growth of *Escherichia coli* with RDX Samples 1E-6E (left to right).

![](https://static.igem.org/mediawiki/2021/5/53/T--RUM-UPRM--tubitos1.JPG)

**Figure 10.** Bacterial Growth of *Escherichia coli* with RDX seen on UV transilluminator with RDX Samples 1C-6C (left to right).

![](https://static.igem.org/mediawiki/2021/1/12/T--RUM-UPRM--tubitos2.png)

**Figure 11.** Bacterial Growth of *Escherichia coli* with RDX seen on UV transilluminator with RDX Samples 1E-6E (left to right).

**### ➜Device 2**

Even though we did not have similar results to those of Device 1, we had one promising result, where Fragment 2 of Device 2 was successfully cloned into the plasmid through the simple cloning method, which is present approximately around the 2.5kb band. In the future, we will attempt to clone fragment 1 and, eventually, the entire device.

![](https://static.igem.org/mediawiki/2021/2/24/T--RUM-UPRM--gelsitod2.png)

**Figure 12.** Gel electrophoresis ran showing the digested Device 2 Fragment 2 present in pUC19 around the 2.5kb area.