iGEM 2018 — Team US_AFRL_CarrollHS

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:US AFRL CarrollHS/Notebook

[![](https://static.igem.org/mediawiki/2018/4/4f/T--US_AFRL_CarrollHS--_WhiteDNALinearLogo.png)](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS)

* Team

  [Team Members](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Team)
  [Collaborations](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Description)
  [Design](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Design)
  [Experiments](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Experiments)
  [Notebook](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Notebook)
  [Interlab](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/InterLab)
  [Model](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
  [Results](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Results)
  [Demonstrate](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Demonstrate)
  [Improve](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Improve)
  [Attributions](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Attributions)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Parts)
  [Composite Parts](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Part_Collection)
* [Safety(current)](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Safety)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Public_Engagement)
* Awards

  [Applied Design](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Applied_Design)
  [Best New Composite Part](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Composite_Part)
  [Best Part Collection](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Part_Collection)
  [Education & Engagement](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Integrated_Human_Practices)
  [Model](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
* [Judging Form(current)](https://igem.org/2018_Judging_Form?team=US_AFRL_CarrollHS)

![Notebook](https://static.igem.org/mediawiki/2018/4/47/T--US_AFRL_CarrollHS--NotebookHeader.jpg)

# Week 1

## Monday (06/04/18)

### Lab 1

The students brainstormed ideas on project goals and ideas. After briefing many ideas, the students and Mike ultimately decided to bring a “sense and attack” approach to the project. In theory, the module would be able to move naturally towards jet fuel biofilms via chemotaxis, and a higher concentration of biofilms would allow the module to swim towards biofilms before attacking the biofilm and nullifying it before it destroys the jet fuel. Researchers and students alike were excited at the idea and eager to begin the research.

### Lab 2

The RX students eagerly began their first day of work by acquiring their base passes early in the morning. They then left to UES where they had a meeting with UES employees and they signed papers and contracts to solidify their positions.
They then arrived on base where they were introduced to the colleagues they would be working with inside of the lab and were shown around the lab. With the assistance of previous year team member, Andrea Poole, they made 2 liters of LB Broth. They also made 2 liters of LB Agar of which they added Ampisilin and used the solution to pour plates.

## Tuesday (06/05/18)

### Lab 1

In an effort to reintroduce the students the lab after the break, they began the day by making LB Media. This was also another way to ingratiate the new members of the team to become familiar with real world lab experience and excite them in pursuing the project. Later, the students spent time researching possible project ideas and ways of utilizing quorum sensing and chemotaxis. They spent the day researching in literature and beginning preliminary wiki work.

### Lab 2

The students sat through a 3 hour long meeting from 9:00 until around 11:30 as a portion of their in-processing into the RX laboratory.
The students in the lab performed a transformation of the plasmid by placing it into the cell. After this was accomplished, the students then plated the LB Agar plates.

## Wednesday (06/06/18)

### Lab 1

The students spent most of the day brainstorming ideas in order to host a meet-up for nearby iGEM teams. At the meeting with their mentor, the students discussed the viability of different “search and attack” mechanisms and current research being done to possibly implement into the project. The team brainstormed and looked into different ways of being able to sense fungi.

### Lab 2

The students of the RX lab picked colonies from the plates that were streaked the day before. These picked colonies were streaked onto a new plate of LB with ampicillin and then flicked into a culture tube filled with LB broth with ampicillin.
After these steps were finished, the students then traveled to UES to research various topics for project ideas

## Thursday (06/07/18)

### Lab 1

The LabPats began the day by making plates with chloramphenicol resistance. The students plated their old *E. coli* cells from last year and letting it settle in incubation. Later in the afternoon, the students and mentors discussed the viability of their project idea and creating a basic plasmid outline to begin sequencing through IDT.

### Lab 2

The students of the RX lab performed a mini-prep of their culture tubes as a way to teach the new members of this process and as a refresher to the previous iGEM students
The students then traveled to UES to continue research each of their various topics.

## Friday (06/08/18)

### Lab 1

Early in the morning the students listened to a presentation given by Vanessa about fungal contamination on aircraft. The students were very excited as they themselves were working with biofilm contamination in aircraft fuel tanks and it was very eye-opening to listen to their own mentors talk about her own research in the area. The students were able to learn a lot and absorbed new information on how to approach their project and design their own plasmid module. Next, the plates with the old colonies from last year were put in the fridge to be grown as a cell culture on Monday. Afterwards the students began introductory MatLab courses on modelling and how to model synthetic biology in order to enhance their project this year.

### Lab 2

The students of the RX lab started their day by attending a mandatory general safety presentation for their inprocessing.
After, they were trained on how to perform a digest of the of the extracted DNA. They next formed a gel and ran the digested DNA on the gel.

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Week 2

## Monday (06/11/18)

### Lab 1

The day began with an early lab meeting. The goal of the meeting was to ingratiate the new members with basic lab rules and procedures. Afterwards, the students prepared a presentation about a current project proposal to present to the other half of the team as well as practicing to a few mentors who had been on vacation. Later in the afternoon, the students grew up the PRhl\_GFP cells from last year and also streaked plates of a “RhlR” plasmid from Dr. Goodson.

### Lab 2

The students, realizing that they made a mistake in the running of the gel, Friday, they performed the digest and then ran another gel of which they took a picture.
The students then proceeded to UES to perform research over each of their own delegated topics

## Tuesday (06/12/18)

### Lab 1

Early the next morning, the students began by performing a mini-prep on the grown up Phrl\_GFP cells in order to retrieve the plasmids. After viewing fluorescence under a UV light, they were able to determine that last year’s glycerol stock solution was still usable. Next, they picked colonies from Dr. Goodson’s “RhlR” in order to begin growing up in the incubator. After a quick lunch, the students headed to the meeting with their mentors and USAFA team in order to give a short briefing about their current project proposal and method of approach.

### Lab 2

The students of the RX lab started off the day with a presentation to their lab from 9-10:30
Then the students then proceeded to UES to meet with the Carroll HS mentors to discuss their project ideas and continue with research

## Wednesday (06/13/18)

### Lab 1

The LabPats began the day by performing a mini-prep on the RhlR cultures in order to extract the plasmid for later transformations and digestions. Before they could continue with their project, the students needed to find primers and utilize the Gibson Assembly in order to continue on with their plasmid design. After a long day in the lab, the students began research on the specifics of the Gibson Assembly and how to design their own primers. Annie also began preliminary Wiki work, Yazmin emailed a professor to acquire a certain *E. coli* strain with the CheZ gene knocked out. Jason continued learning about mathematical modelling.

### Lab 2

The students started off their day by continuing their research into the cinnamaldehyde concept for their portion of the project.
They then met with Chia, Vanessa, Drew, and Andrea in order to present their ideas for the project and receive the critical feedback needed in order to enhance their project and provide a direction to follow.
During the discussion and presentation, an idea was brought up by the mentors to fuse the chitinase enzymes onto the outside layer of the *E. coli* cells while embedding genes in order for the cell to produce the poisonous cinnamaldehyde. This combination was agreed upon by both the mentors and the students to pursue.
The students then attended a safety meeting on base from 1-2.
They then proceeded to travel to UES and there they continues their research on the now 2 decided upon topics.

## Thursday (06/14/18)

### Lab 1

Dr. Breedon assisted the LabPats in designing primers and sequencing their plasmid for IDT to make. They discussed plans on trying to make their own plasmid from scratch via Gibson Assembly, as well as double checking the final product with the IDT construct. They ordered many primers and parts, like different ribosome binding sites attached to the CheZ gene.

### Lab 2

The students in the RX laboratory began their day at UES researching the now solid plan of binding two forms of chitinase to the outside of the *E. coli* cell membrane while having the cinnamaldehyde break into and kill the fungi cells.
Max researched the process of which the cinnamaldehyde kills the cells and the production levels of phenylalanine and cinnamaldehyde plus the lethal concentrations.
Peter and Travis dived into deeper research of the various forms of possible chitinase options for the use of the project. They completed this by making a table focusing on the genome of the chitinase production, the bacteria the genome derives from, optimal growth conditions, impacts on fungi, and the size of the chitinase
The students at the end of the day traveled to RX and meet with Chia to discuss the research they had found and to narrow down the specific research to perform; i.e. the sequences of the genes

## Friday (06/15/18)

### Lab 1

The LabPats met at UES to begin discussing and planning Human Practices.

### Lab 2

The students started on Friday by attending a chemical safety training course presented at the RX building. After this hour and a half safety presentation, they once again met with Chia. Chia gave them a task list of research to do for the day:
Research the ice nucleation fusion protein for the chitinase,
Narrow down which two chitinase genes to use,
and look into the sequences for all of the genes.
Chia also introduced the students to the genome sequence planner named APE.
The students planned to use the three separate components for the formation of the cinnamaldehyde along with two RBS and the promoter (an improvement of an old part) and to use the ice fusion protein with the two forms of chitinase (two new parts to the iGEM part registry)

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# Week 3

## Monday(06/18/18)

### Lab 1

The students began bright and early with their first presentation to the lab. The lab meeting included many prominent scientists from around the department excited to hear about the summer intern’s development with a new iGEM project. The team heard a stellar presentation by USAFA iGEM before giving their own and receiving praise as well. The LabPats are still currently waiting for their sequenced parts and primers to arrive before proceding with further lab work. Thus, they took this opportunity to learn more about wiki development and modelling, with Annie deciding to implement bootstrap into their wiki design.

### Lab 2

On the Monday starting the week, the RX team started their day on base with an RX branch meeting. They then met with Chia and went into further detail of DNA sequencing. After the meeting with Chia, the students went to UES and researched and found and planned the DNA sequencing for both forms of chitinase and the cinnamaldehyde.

## Tuesday (06/19/18)

### Lab 1

Early on the workday, the students took the overnight cultures of the “RhlR” plasmid and tested the fluorescence level to ensure the GFP still worked and that the plasmid wasn’t corrupted. It worked exactly as intended, and the students were able to obtain great fluorescence and pictures. They wanted to have a backup for later just in case, and created a glycerol stock of their “RhlR” plasmid. They then performed a genomic sequence protocol to extract the DNA from the RhlR plasmid so that they can amplify the CheZ gene later when designing the various plasmids.

### Lab 2

On Tuesday morning, the team met at UES where they discussed various points of action that would allow them to accomplish their goal of the cinnamaldehyde and chitinase producing bacteria.
Peter, Chris, and Travis worked on researching as many forms of chitinase as possible and searching for the DNA sequences of each chitinase.
Max began research on the pathways of cinnamaldehyde production and how to piece together to the three genes from the iGEM registry and which ribosomal binding sites to use.

## Wednesday (06/20/18)

### Lab 1

The LabPats went to UES to focus on building the presentation.

### Lab 2

The iGEM students of the RX lab began their day at UES. They checked their work of the previously completed DNA sequences and made adjustments. The entire team including RH then had a skype conversation with the New Deli iGEM team from India. After this conversation, the RX team then proceeded to head to RX in order to meet with Chia.
At RX, Chia taught the students an in-depth method of how they were to use restriction site within their experiments and DNA sequence. He then gave the team further guidance of how to correct their DNA sequences.
Back at UES, the RX team then split up into their various different tasks:
Chris downloaded software onto the UES laptop and worked on the logo.
Peter fixed the DNA sequences of the chitinases and sent them to Chia and then worked on designing the logo.
Travis began work on the safety forms with Yazmin with the help of Tina, Dallas, and Andrea.

## Thursday (06/21/18)

### Lab 1

With DNA sequences still coming in, the students were out of the lab for today. Today they learned a lot about DNA sequencing programs and further modelling methods.

### Lab 2

RX lab students started their morning on base. They first met with Venessa to discuss the plans for the meeting on the following day (Friday, 22nd). Next the team met with Chia in order to finalize their DNA sequences in order to insure correctness so that they may order the genome carrying plasmids from IDT. They made a plan to meet with him later in the day once he finished reviewing their work.
The students then made their way to UES to begin on their various tasks:
Max worked on the cinnamaldehyde sequence and the project description.
Peter worked on the project description and on the team logo.
Chris also worked on the project description and some research on the various bacteria and fungi that will be used in the project for the safety forms.
Travis worked on the daily lab logs, some research on the various bacteria and fungi that will be used in the project for the safety forms, and on the safety forms themselves.
The students then returned to base to meet with Chia and discuss the final submission of the DNA sequences and the iGEM mini-prep. They then decided to start the mini-prep by making SOB and CCMB80.
They then returned to UES to complete the previously stated tasks.

## Friday (06/22/18)

### Lab 1

The students are still currently waiting for DNA sequences to come in. Mentor Dr. Varaljay thought it would be a perfect opportunity to use NCBI Blast as a method of analyzing DNA sequences with proteins and nucleotides. She gave a quick lesson on how to use it and how to discover more about unknown surface proteins pertaining to the project.

### Lab 2

On Friday morning, the whole iGEM team met at UES for the weekly meeting with Vanessa. She taught the team how to use the NCBI web-based program called BLAST. Vanessa helped the team put in the Chitinase C-1 sequence into the program and cross reference it to other amino acid sequences. After that they put unknown nucleotide sequences into the program in order to identify them. After the meeting with Vanessa, RH left for lunch while RX stayed behind to discuss their sequences with Chia. Chia helped Max to edit his sequences for ordering, and after that Peter, Chris, and Max left for lunch. They met with RH and the cadets at El Meson. When they returned to UES, Max left, then Peter and Chris ordered the sequences through IDT.

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Week 4

## Monday (06/25/18)

### Lab 1

The primers finally arrived! The students were ecstatic to be able to begin lab work again. They began by re-suspending the new primers before utilizing them to perform a PCR on Dr. Goodson’s “RhlR” plasmid and CheZ gene. After a gel confirmed the presence of expected band lengths, the team was prepared to tackle the next task of purifying the gel and utilize the PCR product for preliminary construction of the final plasmid. They also hoped to utilize the CheZ gene in many ways by attaching different ribosomal binding sites and testing them all within the iGEM-RhlR plasmid.

### Lab 2

On Monday morning, the RX students entered lab and created 1 Liter of LB Agar in order to pour plates for the start of iGEM Interlab protocol. They added a chloramphenicol resistance to the LB Agar. After this was made, the team then poured plates so that the Interlab Top 10 *E. coli* will grow.

## Tuesday (06/26/18)

### Lab 1

After a very successful PCR yesterday and picture perfect yesterday gel, the LabPats were ready to begin the gel purification step. After the protocol was completed, the team was devastated to discover that the nano drop machine was out of commission and that it would be difficult to measure the concentration of the purified “RhlR”and “CheZ” bands. After jumping through some hoops, the labpats were able to utilize a neighboring lab’s machine and measure the concentration of both accurately. The measured results were a lot less than expected, and Dr. Breedon expressed her worries about a Gibson assembly protocol being successful. They decided to proceed anyways. In addition, the Dr. Breedon also talked to the students about attaching overlaps on the CheZ gene, and performing a PCR on the three ribosomal binding sites.

### Lab 2

The RX Lab students began their day by attending a branch meeting. After the meeting, the students went to their lab to continue work on the iGEM interlab. For this portion of the mini-prep, the students performed a transformation on the DNA samples that iGEM sent them in the kit. After the transformation was complete, the students headed to UES.
At UES, the students began work on the presentation for the Michigan State meetup which will occur on the following Saturday, June 30th.

## Wednesday (06/27/18)

### Lab 1

The students began the morning by redoing a PCR on the “RhlR” plasmid and “CheZ” gene to gather more concentration the second round. They also began their first Gibson Assembly, hoping to ligate the “RhlR” plasmid to the iGEM backbone. Afterwards, the LabPats ran the “RhlR” and “CheZ” on a gel in order to perform a gel extraction. However, they were disappointed to find out that their “RhlR” plasmid did not produce a band on the gel in order to perform a gel extraction. Disappointed but not defeated, they knew that their plasmid worked, though they could’ve messed up on the PCR.

### Lab 2

The students of the RX lab began their day at their lab reviewing the work they performed the previous day. They looked at their cultures and began work to place colonies into culture tubes for further experimentation. They then gave their culture plates to the USAFA iGEM team in order to assist them in their work of their interlab.
The students then headed back to UES in order to prepare for the team meeting that was to occur that afternoon. Once the meeting began, the entire team discussed with Dr. Carter about events and progress that had occured the week before.
Discussion points included:
Logo,
spirit wear,
safety forms,
mini-prep,
USAFA, and the
Michigan State Meetup.
After the discussion the students practiced their presentation for the Michigan State Meetup and then worked to improve the presentation.

## Thursday (06/28/18)

### Lab 1

After yesterday’s failed attempt at PCRing the RhlR and CheZ bands correctly, the students arrived in the lab with a new desire to accomplish what they didn’t yesterday. They meticulously followed the protocol, taking extra care this time to ensure everything was right. After gel electrophoresis, they were ecstatic to find out all of their PCR’s had gone according to plan and they were ready to perform a Gibson Assembly tomorrow. Even though the Gibson Assembly from Monday (with RhlR and iGEM backbone) produced a shockingly low concentration, Amy decided to have the students attempt to grow up the cells in DH5a on Chloramphenicol plates anyways.

### Lab 2

The RX team started their day on base where they began work on the calibration portion of the iGEM mini-prep. The team split up with Max and Jonah to continue work on the interlab while Chris and Travis went to UES to work on the presentation and finishing the safety forms.

## Friday (06/29/18)

### Lab 1

After a week of solid work, the LabPats were a little caught off guard when they realized there wouldn’t be much lab work today. It being the end of the work week and the lab closing on weekends, the students were not able to do overnights or anything requiring an extra day. Thus, the students started and finished the day checking to see if their “iGEM-Rhl” plasmid with the iGEM backbone grew up. There’s growth!” the students exclaimed as they opened their incubators and examined their plates. As it turns out, there was growth on the “iGEM-RhlR” plates with chloramphenicol. The students knew for sure that their plasmid had been transformed correctly into the DH5a cells. Afterwards, the student had a quick meeting and final tune-up presentation before the Michigan State Meet-up.

### Lab 2

The iGEM RX lab team began their day at UES where they continued to edit the presentation for the Michigan State meet up. They also continuously practiced their presentation and their speaking abilities while the others gave helpful tips and suggestions.

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# Week 5

## Monday (07/02/18)

### Lab 1

It was another short day in the lab as the LabPat’s main focus today was just to grow up cultures of the “iGEM-RhlR” plasmid in DH5a cells. Their goal was to grow it up overnight, then perform a mini-prep to extract the plasmid in order to send it off for sequencing. After the labwork, the students contacted teams at University of Texas for collaboration, Wilbert from National University of Singapore for modelling, and the London School of Boys for Wiki Tips and Tricks.

### Lab 2

To begin the week, the RX lab students began their day at UES. At UES, they reviewed with Dr. Carter the presentation from the previous Saturday at the Michigan State Meetup. At UES, they worked on improving the presentation and made a list of companies to start contacting as a means of aiding in the human practices category. They then began to contact companies through phone calls, voicemails, and emails.

## Tuesday (07/03/18)

### Lab 1

After performing the mini-prep, the nano-drop machine displayed a large concentration of DNA. They sent the “iGEM-RhlR” plasmid off for sequencing, with fingers crossed on achieving the desired sequence. Afterwards, a weekly meeting with the entire team was productive and informative, as everyone reset goals and reassigned tasks to finish the rest of the year out strong.

### Lab 2

The RX lab students began their day by starting the mini-prep of the Interlab samples. They made 64 samples that were to be ran on an agarose gel within the following days.
After the mini-prep was completed, the team headed to UES where they were to have an entire team meeting including the mentors where they discussed:
Michigan State meet up,
spirit wear,
logo,
field trips,
team bonding, and
human outreach.
After the meeting, the team remained at UES where they delegated the various tasks of the summer and began work on each of these tasks.

## Wednesday (07/04/18)

### Lab 1

National holiday today. Off work.

### Lab 2

National holiday today. Off work.

## Thursday (07/05/18)

### Lab 1

The LabPats worked on logo design and the abstract as they waited for sequencing results to arrive. Today was the last day for the USAFA team, and the LabPats decided to go out for lunch to treat them before they left. After a tearful goodbye, the students were hopeful to see them later at the Jamboree. They reached out to MIT labs where they had been experimenting with biofilms within the human body. The sequencing results arrived later and Dr. Breedon explained that the backbone had most likely ligated to itself, allowing the cells to grow but not producing the desired results. The LabPats learn the hard way that not everything in SynBio always goes exactly as planned.

### Lab 2

The RX Lab Students began their day at base where they split into their two groups in order to focus on their two sides of their projects:
Max and Jonah ran Interlab Gel and
began to grow colonies of DH5-Alpha in order to begin testing of cinnamaldehyde.
Chris, Peter, and Travis performed a
Mini-prep of pwPCH055 c.1 and the running of it on a gel, took a picture of it, and nano-dropped the undigested pwPCH055 c.1 DNA.

## Friday (07/06/18)

### Lab 1

Early the next day, the LabPats were eager to re-do everything. They first performed the same Gibson Assmbly, ligating the iGEM backbone with the RhlR plasmid before transforming the product within DH5a. Afterwards, they replated the cells and hoped that favorable results would come Monday.

### Lab 2

To end the week, the RX lab students began their day at the lab where they once again split into the two research and testing groups. Jonah began testing on the grown DH5 Alpha cells from the previous day. He placed various levels of cinnamaldehyde into the DH5 Alpha in order to view its effects on the formed biofilm. Travis, Peter, and Chris performed a gel purification of the chitinase plasmid in order to isolate, amplify, and purify the desired band (removing the tyrosine) .

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Week 6

## Monday (07/09/18)

### Lab 1

IT WORKED!!! Annie exclaimed as she retrieved the plates from the incubator. The plasmid had grown up exactly as planned. They then proceeded and extracted the plasmid from the host DH5a in order to send it in for sequencing.

### Lab 2

On Monday, July 9th, the RX students began work in the lab once again splitting into the two groups in order focus on each aspect of the project.
Jonah took the cinnamaldehyde cultures from the previous Friday and began the process of staining the well plate with crystal violet in order for the amount of biofilm to be measured. The process requires the well plate to dry overnight before any measurements can be taken so it will be continued the next day.
Travis, Chris, and Peter worked on a second digest of the Chitinase Plasmid in order to collect more purified DNA for when the IDT order arrives.
They also used the Nano-drop to test the concentration levels of the undigested DNA and took the data in their laboratory notebook.

## Tuesday (07/10/18)

### Lab 1

The sequencing results arrived early in the day, and the students were both excited and anxious to have a peek at the results. The last time, the results were unfavorable, but this time, with everything going right, the LabPats opened the sequencing document and examined the results. Almost every base pair matched up! It was a huge win and morale booster for the team, as now the students could begin putting the “CheZ” insert into the plasmid finally.

### Lab 2

To begin the day, the entire team met as UES with TIna Davis for a discussion about upcoming due-dates and deadlines. The whole team decided to start on human practice ideas and beginning to brainstorm ideas. Some of these discussiontopics included:
Introduction of the open forum idea at Carroll High School, for which Travis was put on task for the day on making a flyer to hand out at the Carroll High School schedule pickup day; grade school presentation, which Jonah was tasked with; music video, for which Chris and Peter began deciding on a song and writing the lyrics; and Attributions for RX, which was delegated to Max.
They also put on their calendars of every due date in the future and when to have completed items to be sent in for base clearance
Each of the students began work on their designated tasks. Afterwards, the RX students met their colleagues from their lab for a lab outing and bonding.
After the lab bonding, the team went to base to met and talk with Chia about their cancelled IDT orders. With the assistance of Chia, the team fixed their sequences and split the cinnamaldehyde order into 3 parts instead of two.
They also discussed the plan for the next couple of days in lab involving the Interlab.

## Wednesday (07/11/18)

### Lab 1

Another big component of the project would be to test the motility of the cell. By testing cells with the CheZ knockout strain and ones without, the students can observe the motility of CheZ. The cells with the CheZ knocked out doesn’t move and grows a single colony, while the ones with the CheZ should be able to expand and grow many colonies all around the epicenter.

### Lab 2

The RX students began their day in lab where they split into the two research groups.
After collecting the digested Chitinase Plasmid DNA from Monday, Peter, Chris, and Travis created a 6 well agarose gel to run the digested DNA on. After running the gel, the students then performed a Gel Purification which isolated the desired Ice nucleation protein from the tyrosine
They then Nano-Dropped 2 purified DNA samples that they had collected
Max and Jonah completed the staining process of the biofilms treated with different concentrations of cinnamaldehyde. They also began the process of treating the 96 well plate with Isopropanol in order to cleanse it for the experimentation
After completing the lab work, the team headed to UES where they each continued work on their delegated tasks from the previous day. Peter and Chris decided to make the song Lose Yourself by Eminem. Travis completed the flyer.
Jonah continued to update and add information to the grade school presentation

## Thursday (07/12/18)

### Lab 1

At another early morning start, the Labpats began by digesting yesterday’s PCR. Next, they looked at the plates from the motility test, and were pleasantly surprised to see that they looked as expected and grew nicely. They were also able to conclude that the toothpick method worked a lot better than the drop method, as the motility was clearly shown by the expanding non-motile colonies. Aftwards, they made a gel for the PCR digest, ran the gel, and cut the bands. Finally, the students grew up the backbone-RhlR from yesterday’s plates.

### Lab 2

On Thursday the 12th, the RX lab students commenced their day by heading to base where they began work on multiple projects.
Jonah and Max grew up culture to use for the cinnamaldehyde testing which would occur the next day.
Peter, Chris, and Travis took the iGEM Interlab liquid culture samples and worked to patch all 32 samples and freezing down 8 of the samples in the -80 oC freezer (1 from the NEG Control, 1 from POS Control, and 1 from 1, 2, 3, 4, 5, and 6).
A portion of this frozen down culture will be used for the following day as part of the Interlab protocol.
The team then headed to UES where they discovered that a few of the biofuel companies that Chris had contacted had emailed back with information.
One wanted to have a conference call with the team, and
another wanted to publish an article about the iGEM team, which Jason offered to write. The students emailed the companies back to try set dates for these appointments.
They also continued work writing the lyrics.

## Friday (07/13/18)

### Lab 1

Starting off, the LabPats completed a gel purification from the bands that were cut yesterday. The C4 test did not work as expected but the students deduced that it may have been too low of a concentration. Thus, the LabPats chose to re-do it, however; this time with the goal od getting a high concentration. To end off the week, they set up a Gibson Assembly with backbone-RhlR (everything in the plasmid except GFP) with CheZ to create our CheZ plasmid. The students were ecastatic, as the completion of this plasmid would mean they would be almost completely done with their project.

### Lab 2

As the week ended, the RX Lab Students decided to try a practice run of the plate reading of the iGEM Interlab as to hasten the process for when the real data collection was to begin. To begin the protocol, Peter and Max ran an OD readings of the prepared culture while Jonah taught Chris and Travis how to stain the 96 well plates for the biofilm data collection. After the OD readings were recorded, Max and Travis stayed at the lab to finish the protocol while Peter, Jonah, and Chris left for UES in order to attend Vanessa’s bioinformatics and mathematical modeling lesson. After the cell reading Interlab protocol was completed, Max and Travis headed to UES where they met up with the rest of the team and worked on the following tasks: Chris and Peter continued working on song lyrics, Chris contacted companies, Peter worked on Chia the Chitinase and Mike the microbe, Travis began research on the resuspension protocol, concentration, and storage information for the chitinase enzyme, and Max and Jonah did more research on Cinnamaldehyde and the collaborations with fellow iGEM teams.
At the end of the day, Peter and Max returned to lab to finish the 6 hour Interlab protocol and collect preliminary data

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# Week 7

## Monday (07/16/18)

### Lab 1

Another day closer to finishing their project! The LabPats arrived at the lab with the goal of being able to send their finished RhlR-iGEM plasmid with the CheZ in for sequencing. However, after checking the plates in morning, the LabPats were disappointed to find that the plates did not grow at all. Nevertheless, they decided to re-do the PCR, Gel Electrophoresis, Gel Extraction, and transformation again. After another long day, the students were able to complete the PCR, Gel Electrophoresis, and stored the gel in the fridge.

### Lab 2

To begin the week, the RX Lab students split into the two groups as to each focus on each aspect. Travis, Peter, and Chris realized after meeting with Chia that an extra KPN1 site was present within their plasmid which was cutting out the extra 20 base pairs needed to assembly, so, on Sunday, Chia came in and made clones of the pwPCH055 plasmid with the extra KPN1 site removed. The students then patched and inoculated the plasmid culture.
Max and Jonah continued work on performing experiments to discover the effective concentration of cinnamaldehyde on bacteria.
After the lab work was completed on base, the students then headed to UES where they continued working on the human outreaches to companies, the song lyrics, and the mike the microbe collaboration.

## Tuesday (07/17/18)

### Lab 1

The day was hectic yet again. They checked their overnight cultures and discovered that no gFP had been expressed. For some reason, the bands on the PCR displayed a continuous long band down the middle instead of a clearly defined, horizontal band. The students were dismayed and disappointed, realizing all their work from yesterday was for nothing. However, they retraced their steps back to square one. At the beginning of their project, they had tested Dr. Goodson’s “RhlR” plasmid that clearly worked and decided to go back to the glycerol stocks they had come from and be able to regrow out the cells to work with them. They were able to prep overnight cultures and streaked them out on the plates. Meanwhile, the LabPats also wanted to re-do the PCR once again of the overnight cultures just to make sure that they hadn’t just been things wrong the days prior. They discovered, yet again, that the singular band had not shown up but instead a long band ran down the length of the gel. There was also a team meeting later in the day that the entire team congregated at to discuss project progress as well as possible human outreach “field trips” to the Cleveland Becket Oil and an oil subsidiary of Marathon in Cincinnati.

### Lab 2

At the start of the morning, the RX lab students went to lab where they split into the two teams to focus on each side of the destroy aspect of the project.
Peter, Travis, and Chris performed a mini-prep, digest, and ran a gel of the KPN1 removed pwPCH055 plasmid. After viewing the ran gel, 5 colonies were to be good clones with the KPN1 site removed and they were chosen for further culturing and experimentation.
Max and Jonah continued their work on the serial dilutions of chitinase testing against Yarrowia and Nissile in order to collect data on an effective concentration of cinnamaldehyde .
After the lab work was completed, the RX students met at UES with all of the mentors for an all team meeting where they discussed project progress as well as possible human outreach “field trips” to the Cleveland Becket Oil and an oil subsidiary of Marathon in Cincinnati.

## Wednesday (07/18/18)

### Lab 1

Today’s plan included a digestion protocol, purification, and ligation on the IDT order with the with experience after doing so many for so long. They were able to complete everything and then head off to the team photo shoot at the United States Air Force Museum, where they took team pictures in front of many iconic planes.

### Lab 2

The RX lab team started the day once again in lab where they discussed the direction their project was heading. Max and Jonah began discussion since their cinnamaldehyde parts were cancelled by IDT along with the Chitinase B4A part. The team reexamined the DNA sequences and found an error in one of their parts. They fixed the error and split the two cinnamaldehyde parts into 3 parts instead of two.
With this dilema out of the way and the new parts ordered, the team headed to the lab where they once again split into two working groups.
Peter, Travis, and Chris spent the rest of the day performing a digest of the KPN1 removed plasmid DNA and ran a gel with it. After the gel was ran and a picture was taken, a gel extraction was performed. The final concentration of the extracted plasmid was 7.9 ng/uL which seemed to be low but acceptable for the DNA assembly and transformation that was planned to occur in the following days.
Max and Jonah set up an experiment where they isolated colonies on plates and performed a serial dilution on the plate of cinnamaldehyde.

## Thursday (07/19/18)

### Lab 1

The students were ready for another busy day in the lab! They discussed with mentors about the status of their project, and decided to proceed with using IDT parts in order to ligate their RhlR Insert, iGEM backbone together in order to perform a transformation and grow them up in DH5a cells. In addition, they were tasked with performing another Gibson assembly, this time joining the CheZ inserts with various ribosomal binding sites to the insert and backbone. Both transformations went into the incubator overnight, with the LabPats hoping for its success. Late in the day, Annie performed the Flow Cytometry portion of the Interlab.

### Lab 2

The RX lab students woke up very early in the morning with a plan to collect Interlab data. They started by taking the prepped Interlab cultures and taking the OD reading in order to determine how much of the sample was needed in order to reduce the ABS 600 to 0.02 in a 12 mL culture. A time 0 samples were placed on ice and the time 6 were placed in the incubator as according to the protocol.
After Interlab was finally set up, the team split into the two groups once again in order to cover more ground and complete the necessary work of the project.
Peter, Chris, and Travis started testing on Yarrowia covered plates with various concentrations of chitinase from Streptomyces Griseus in order to find the effective concentration. They also resuspended Chitinase C-1 from IDT and began assembly of the chitinase into the ice nucleation plasmid. After the assembly was completed, they then transformed onto Amp plates and left to incubate overnight and hopefully grow colonies.
Max and Jonah continued with the plate testing of cinnamaldehyde since this is a multiple day process with a lot of waiting periods for growth. They also took the readings of the Interlab samples after the 6 hour incubation period was completed and then sent the samples over to RH for the flow cytometry readings.

## Friday (07/20/18)

### Lab 1

The next day, the LabPats looked at the transformations from the previous day. They were disappointed to see that none of the transformations worked. Saddened, they continued to persist and discuss new ways to approach it. First, they settled on re-doing the PCR, as there may have been an error early on in the process before extracting the “RhlR” plasmid. Thus, they started again, completing a PCR and running it on a gel. After looking at the bands, however; the students were still disappointed to discover that the bands were popping up in the wrong place and the backbone, which is supposed to be at 3,000 base pairs, did not appear as so. Looks like it’s back to the drawing board!

### Lab 2

As the week came to an end, the RX lab students began their morning by eagerly viewing the transformation plates and the Yarrowia testing plates from the previous day. The Yarrowia and chitinase testing plate showed a very visible zone of clearing where the 10 mg/mL and the 1 mg/mL concentrations were tested. Unfortunately, the transformation plates did not grow a single colony after the incubation period.
The lab work for the day included a second Yarrowia testing as a attempt to recreate the results from the first testing and a second DNA assembly and transformation for chitinase C-1 and the pwPCH055 plasmid. Also, in a disappointing turn of events, Max and Jonah’s cinnamaldehyde testing plates were viewed to be contaminated and had to be thrown away, so new cultures and testing was started.

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Week 8

## Monday (07/23/18)

### Lab 1

After discussing all of their options, they discussed ways their PCR might have messed up. Thus, they began by double checking their primers, before agreeing that it was indeed correct. Next, they concluded that there may have been a problem with the digest that was performed, as there were multiple cut sites for the DpN1 digest that may have messed up the bands from cutting in the right places. Armed with this knowledge, Mike suggested them re-doing the PCR, this time leaving the digest out and see if there are the right bands.

### Lab 2

To begin this Monday in lab, the RX Lab students first looked at the Yarrowia Testing plates from the Friday before. Not a single plate seemed affected by the chitinase, so the team made a plan to start another round of testing the following day once the another Yarrowia culture was grown.
The team then split into the two working groups:
Max and Jonah resuspended their IDT orders for the cinnamaldehyde and began a DNA assembly.
Travis, Peter, and Chris performed a DNA assembly transformation on Chitinase C-1 which was set up in an incubator to grow overnight.
After the lab work for day was completed, the RX students met with the RH students in order to work on completing the presentation for Dr. Niak.

## Tuesday (07/24/18)

### Lab 1

The LabPats began another PCR this time with the exact same backbone and primers as yesterday, except skipping the Digestion step with DpN1 directly after PCR completion. Unfortunately, after a full day of diligently performing the PCR and running the gel, the correct band was still missing from the final gel. The students were visibly upset, but very ready to continue to persist until the desired result was achieved.

### Lab 2

To start off the Tuesday, the team viewed their DNA transformation of Chitinase C-1 and to their surprise, no colonies grew. A plan was made to redo the transformation and redo the DNA assembly.
The team then split into the two working groups:
Max and Jonah transformed the assembled cinnamaldehyde DNA and performed another round of cinnamaldehyde testing.
Travis, Peter, and Chris, as stated earlier, performed the 2nd DNA transformation and assembled for a second time in the hopes of obtaining a higher DNA concentration. They also started another round of Yarrowia Testing.

## Wednesday (07/25/18)

### Lab 1

Today the LabPats tried a new PCR method, using gradient temperatures to test and see if the PCR would work and the primers would be able to bind using different temperatures. Unfortunately, it did not work again, and the LabPats decided that there may have been a problem with the primers. They reviewed their suspicions with their mentors and Mike confirmed that they may need new primers.

### Lab 2

The RX lab students entered lab with high hopes. They first viewed the two transformations, one Chitinase C-1 and one Cinnamaldehyde, but both transformations had failed. Once the students talked with their mentor, Drew, they decided on carry out two plans.
The team then split into the two working groups.
Max and Jonah decided to clone the bacteria containing the iGEM Backbone Plasmid in order to increase concentrations of the vector so hopefully it will accept the insert.
Travis, Peter, and Chris took the 2nd assembled DNA which had a higher concentration and transformed it into 3 cells: NEB 5-Alpha, One Shot Top 10, and One Shot Mach 1, in the hopes that one of the three would produce viable colonies. They also set up Yarrowia Testing #4 after discovering that Yarrowia Testing #3 once again showed no results.

## Thursday (07/26/18)

### Lab 1

The LabPats re-completed the Interlab after realizing that they were missing a set of wells from the last time. They spent the day completing the CFU protocol, working with the plate reader, and calibrating the flow cytometer for use. Later in the day, Mike helped the students locate primers and sequences in order to order more primers in time. After a long day with the Interlab, the students were happy to proceed forward with their project.

### Lab 2

With all of the failed transformations in the week, moral was low for the RX Lab Students. They entered lab and found a surprise, the Chitinase C-1 transformations were successful in 2 of the 3 bacteria strains!
The team then split into the two working groups.
Max and Jonah cultured the transformed plasmid of the iGEM Backbone.
Travis, Peter, and Chris cultured the chitinase C-1 transformed bacteria and set up Yarrowia Testing #5 after Yarrowia Testing #4 once again showed no viable results.
After the lab work was completed, the team headed to UES where they completed their presentation and went through a dry run of it in order to prepare for Dr. Naik the following day.

## Friday (07/27/18)

### Lab 1

Stress and nerves were high in the morning when the entire team met in order to present their summer project to the chief scientist, Dr. Naik. The presentation went smoothly and Dr. Naik loved the work! The team celebrated by going out to lunch then they headed back to lab where they began work.

### Lab 2

Stress and nerves were high in the morning when the entire team met in order to present their summer project to the chief scientist, Dr. Naik. The presentation went smoothly and Dr. Naik loved the work! The team celebrated by going out to lunch then they headed back to lab where they began work.
The team then split into the two working groups.
Max and Jonah mini-prepped, digest, ran on an agarose gel, and extracted the plasmid vector of the iGEM backbone needed for assembly.
Travis, Peter, and Chris mini-prepped, digested, and ran on an agarose gel 4 clones from transformed Chitinase C-1. The picture of the gel revealed that the chitinase was cut in half with the enzymes used, so they planned to test in order to discover what happened next week.

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# Week 9

## Monday (07/30/18)

### Lab 1

The LabPats attempted their PCR again with no luck.

### Lab 2

To begin the week in lab, the RX Lab Students planned their goals for the week. They wanted to discover why the chitinase was cut during digestion and once that problem was figured out, then assemble pPTCC1 (Transformed Chitinase C-1). They also wanted to try to test a combination of cinnamaldehyde and chitinase on Yarrowia and on Byssochlamys. Finally, they wanted to successfully assemble cinnamaldehyde into the iGEM backbone.
The team then split into the two working groups. Max and Jonah began another DNA assembly of cinnamaldehyde in order to obtain a higher DNA concentration. They also began Yarrowia and Byssochlamys culture. Travis, Peter, and Chris started an enzyme test in order to discover whether it was a bad enzyme making the cut. Finally, they mini-prepped and digested 20 colones (10 from NEB 5-Alpha and 10 from One Shot Mach 1) that Chia had prepared culture for the previous day. Each clone showed that the chitinase C-1 was being cut in half.

## Tuesday (07/31/18)

### Lab 1

The LabPats attempted their PCR again with no luck.

### Lab 2

On the second day of the week, the RX Lab Students kept working towards the weeks goals.
The team then split into the two working groups. Max and Jonah began Byssochlamys testing. Once they found that their transformation failed again, they decided to order PCR primers in order to PCR the cinnamaldehyde. Travis, Peter, and Chris ran on an agarose gel the 20 colones (10 from NEB 5-Alpha and 10 from One Shot Mach 1) that had been digested the previous day. Each clone showed that the chitinase C-1 was being cut in half.

## Wednesday (08/01/18)

### Lab 1

The LabPats attempted their PCR again with no luck.

### Lab 2

The team entered lab where they kept working towards the weeks goal, some of which were updated and changed.
The team then split into the two working groups.
Max and Jonah tested a combination of cinnamaldehyde and chitinase against plates containing Yarrowia. After the testing was set up, they headed to UES in order to begin Wiki Content.
Travis, Peter, and Chris performed a digest and ran a gel on the pPTCC1 in order to discover what was cutting the chitinase in half. The gel revealed there was an extra BamH1 site that was cutting the chitinase. This is good news since this can be worked with for future experimentation. They also resuspended the IDT order of Chitinase B4A and assembled it with the ice-nucleation protein.

## Thursday (08/02/18)

### Lab 1

The LabPats attempted their PCR again with no luck.

### Lab 2

The team split into the two working groups.
Max and Jonah viewed the Yarrowia testing with chitinase and cinnamaldehyde and took pictures of the results. They then set up another round of testing. Afterwards, they headed to UES to continue work on Wiki content.
Travis, Peter, and Chris received PCR primers for the ice-nucleation protein and the two chitinases. They resuspended the primers and set up a PCR reaction. They also began a transformation of the B4A Chitinase.

## Friday (08/03/18)

### Lab 1

After various unsuccessful attempts with their PCR, the LabPats decided to order their construct, PRhl\_31\_CheZ\_J23117\_34\_RhlR from IDT.

### Lab 2

With the week not working out as they had hoped, the team remained positive for the day and the following, final week of summer in the lab.
The team then split into the two working groups.
Max and Jonah headed straight for UES in order to work on Wiki content
Travis, Peter, and Chris sent in the Chitinase C-1 in for sequencing in order to make sure that a mutation had not formed. They also saw that the Chitinase B4A Transformation had failed. They then headed to UES to also work on Wiki Content.

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# August, September, and October

## The Lab Pats returned to school, so they were not working everyday in the lab anymore. However, the Lab Pats continued their research periodically. In summary:

### Lab 1

During the months of August, September, and October the LabPats focused on repeating the ligation and transformation of the iGEM backbone, and our PRhl\_31\_CheZ\_J23117\_34\_RhlR ordered from IDT. Finally, after a successful ligation, the LabPats transformed their ligation into DH5a cells, plated the transformation, and left it overnight. Afterward, a colony PCR was run to confirm that our transformation was successful. Overnight samples were made from transformation, and miniprepped. The miniprep was dried down in order to submit and ship to iGEM. The miniprep was digested and then ligated into CheZ knockout competent cells that were made from the *E.coli* strain UU2685 in order to perform motility. Ligation was then transformed and plated. Colonies were present on plate and a colony PCR was ran to confirm insert. Due to unexpected difficulties, we have not been able to perform the motility testing that we planned, but look forward to continuing testing.

### Lab 2

Due to the incorrect sequencing of the original primers, new PCR Primers were ordered in order to amplify the DNA of the Chitinases and Cinnamaldehyde for assembly. These orders were patiently awaited all month. While waiting on the primers, the entire team worked on the wiki, the presentation for Jamboree, and began work on designing the poster. Late in September, the PCR Primers finally arrived and work resumed. The chitinases and the cinnamaldehyde were amplified and many assemblies and transformations were attempted, but not succeeded. Throughout the entire lab process, high concentrations of the iGEM vector plasmid were extracted and new protocols for assembly were attempted.Outside of the lab work, the team focused this month on fine-tuning their presentation and creating a background story for the presentation. Mock presentations were made to the school and mentors and criticism was taken and used to improve the presentation. Several students of the team also presented a basic lesson of synthetic biology and our project to local grade schools as part of human outreach. Finally, the team also worked on creating wiki content and on the poster for Jamboree.We got two parts! Weeks of hard lab work finally paid off after the transformation and cloning of the bacteria containing our plasmid assembled into the iGEM backbone was accepted. Chitinase C-1 and Chitinase B4A were then dried down and sent to iGEM.This month was spent fine-tuning the presentation and poster. Wiki is being completed and the team met with our local government representative, Mike Turner, to discuss the future of synthetic biology and legislation of biodiesel. It is a race to get everything perfected before traveling to Boston, but we are all excited for the trip and the challenge!

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Contact us @iGEMLabPats or iGEM@carrollhs.org


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# Team:US AFRL CarrollHS/Experiments

[![](https://static.igem.org/mediawiki/2018/4/4f/T--US_AFRL_CarrollHS--_WhiteDNALinearLogo.png)](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS)

* Team

  [Team Members](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Team)
  [Collaborations](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Description)
  [Design](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Design)
  [Experiments](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Experiments)
  [Notebook](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Notebook)
  [Interlab](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/InterLab)
  [Model](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
  [Results](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Results)
  [Demonstrate](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Demonstrate)
  [Improve](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Improve)
  [Attributions](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Attributions)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Parts)
  [Composite Parts](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Part_Collection)
* [Safety(current)](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Safety)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Public_Engagement)
* Awards

  [Applied Design](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Applied_Design)
  [Best New Composite Part](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Composite_Part)
  [Best Part Collection](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Part_Collection)
  [Education & Engagement](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Integrated_Human_Practices)
  [Model](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
* [Judging Form(current)](https://igem.org/2018_Judging_Form?team=US_AFRL_CarrollHS)

![Experiments](https://static.igem.org/mediawiki/2018/3/30/T--US_AFRL_CarrollHS--ExperimentsHeader.jpg)

# Transformations

## BI21 Cells:

- Thaw 50 µL vial of BL21 cells on ice

- Inoculate 2µL DNA into 50µL BL21 cells

* incubate on ice for 30 minutes

* heat shock at 42℃ for 10 seconds

* incubate on ice for 5m

- Inoculate 250µL LB into vial

* incubate at 37℃ with shaking for 1.5 hours

- Plate 1x/9x

## JM109 Cells:

- Thaw vial of JM109 cells on ice

- Label 14 mL Falcon tube & plane on ice

- Inoculate 50 µL JM109 cells into Falcon tube

- Inoculate 2µL DNA into 50µL JM109 cells

* incubate on ice for 20m

* heat shock at 42℃ for 45 seconds

- Inoculate 950µL LB into vial

* incubate at 37℃ with shaking for 1.5 hours

- Plate 1x/9x

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Electrophoresis/Gel Protocols

## 1% Agarose Electrophoresis Gel

- Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask

- Heat in microwave for 35 seconds

- Swirl and heat in microwave for 25 seconds

- Swirl and heat in microwave for 15 seconds

- Swirl and heat in microwave for 7 seconds

- Swirl and ensure no ‘floaties’

- Cool outside of conical flask with water until ‘hand not’

- Pour into gel mold and add well comb

## Gel Extraction

- Use ethanol-wiped scalpel to excise appropriate bands

- Mix and incubate at 50°C for 10 min

* Vortex every 2 mins until gel slice is completely dissolved

- Place nucleospin column into 2mL collection tube

* Add sample

* Centrifuge at 11,000 xg for 1 minute

* Discard throughflow and place column back in same tube

- Add 700 mL Buffer NT3

* Centrifuge at 11,000 xg for 1 minute

* Discard throughflow and place column back in same tube

- Repeat step 4

- Centrifuge at 11,000 xg for another minute

* To remove buffer

+ Discard throughflow

+ Centrifuge at 11,00 xg for add minute

+ Place column in a clean, labelled 1.5 mL tube

- Add 25 mL prewarmed (50°C) Buffer NE

* Incubate at 50°C for 5 min

* Centrifuge at 50 xg for 1 minute

* Centrifuge at 11,000 xg for 1 minute

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# Plasmid Transformation Protocol

- Aliquot 1 ml media into 1.5 mL tubes

* Place in 42°C waterbath

- Prechill labelled 14 mL round-bottom falcon tubes

* Add mL cell culture to tube

* Add appropriate amount of ligase rxh (2-2.5 L)

* Incubate in nice for 20 minutes

- Heat shock cells by placing at 42℃ for 45 seconds in waterbath

* Place on ice for 2 minutes

- Add 950 mL preheated media to each tube

* Incubate at 37℃ with 215 rpm shaking for 1.5 hr -> 20 minutes

## Plating

- Plate on LB-antibiotic plates

* Plate 100 ml for 1x transformation

* Plate 100 mL 9x centrifuge remaining transformation at 2000 xg for 5 minutes and resuspended pellet in 100 L media

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Plates Protocol

- Follow LB Agar Recipe

* Make sure to put stirring bar in water

* Needed to make into homogenous gel

* Needs to be sterile

- Put into Autoclave

* Lid should not be too tight

- Put in ice bath 42°C

* Cool till not burning hand

* Put on stirring plate

- Put sterilized plates in Lamenia shield

- Using micropipette put .5 mL of chlor. And amp. agar solution

* 1000x dilution

* Amp. has to be defrosted in dark drawer

- Pour solution into plates

* Thin layer fill bottom

* Take lid slightly off to prevent condensation

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# QIA Prep Spin Mini Prep Kit

- Example for 18 mL LB add

* 18 L20 mg/ L Kanamycin (antibiotic)

* 18 L 50 mg/ L ampicillin

- Aliquot 3.5 mL into labelled round bottom falcon tubes

- Using sterile toothpicks- pick a single colony forming unit (cfu) and inoculate tube

* Incubate overnight at 37°C with 215 rpm shaking

- To 100 mL 60% glycerol stock, add 300mL overnight culture to appropriately labelled tube

* Store at -80°C

- Pellet remaining cells by centrifugation in 1.5 mL tube at 13,2000 rpm for 1 min

* Discard supernatant and repeat

- Resuspend pellet in Buffer P1 250 mL

* Add 250 L Buffer, P2 and mix by inverting 10x

* Add 350 L Buffer N3 and immediately mix by inverting 10x

- Centrifuge at 13, 200 rpm for 10 minute

- Pipet supernatant into labelled QIA prep spin column

* Centrifuge at 13, 200 rpm for 1 minute

* Discard thoroughflow

- Wash column by adding .5 mL Buffer PB \*regular for low copy number plasmids\*

* Centrifuge at 13, 200 rpm for 1 minute

* Discard thoroughflow

- Wash by adding .75 mL Buffer PE

* Centrifuge at 13, 200 rpm for 1 minute

* Discard thoroughflow

* Centrifuge for an addition minute to ensure removal of residual wash buffer

- Place spin column in a clean, labelled 1.5 mL tube

* Add 50 LEB to center of column & let stand for 1 minute at room temperature

* Centrifuge at 12,000 rpm for 1 minute

* Store at -20℃

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Making Motility Plates

- Follow Motility Plate Recipe

* Recipe

+ 5 grams of Tryptone

+ 2.5 grams of NaCl

+ 1.25 grams of Agar

+ 500 mL H2O

* Make sure to put stirring bar in water

* Needed to make into homogenous gel

* Needs to be sterile

- Put into Autoclave

* Lid should not be too tight

- Put in water bath 42°C

* Cool till not burning hand

* Put on stirring plate

- Put sterilized plates in Lamenia shield

- Pour solution into plates

* Thin layer fill bottom

* Take lid slightly off to prevent condensation

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# Making TSA Plates

- Follow TSA Plate Recipe

* Recipe

+ 30 g of TSB

+ 15 g of Agar

+ 1 Liter of H20 18 mΩ

* Make sure to put stirring bar in water

* Needed to make into homogenous gel

* Needs to be sterile

- Put into Autoclave

* Lid should not be too tight

- Put in water bath 42°C

* Cool till not burning hand

* Put on stirring plate

- Put sterilized plates in Lamenia shield

- Pour solution into plates

* Thin layer fill bottom

* Take lid slightly off to prevent condensation

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Yarrowia Testing

## Growing Yarrowia Culture

- Pipette 5 mL of TSB into a culture tube

- Use a wand to swipe a colony of Yarrowia from an already grown plate of Yarrowia

- Place wand with colony of Yarrowia into culture tube

* Flick wand for 15 seconds

- Incubate overnight at 27°C with 200 rpm shaking

## Preparing Chitinase Testing Enzyme

- Measure out 2 g of chitinase from Streptomyces Griseus

* Place into 2 mL microcentrifuge tube

- Resuspend chitinase

* Pipette 0.100 mL of PBS into tube

* Vortex tube for 15 seconds

- Store in -20°C freezer

## Chitinase Testing Against Yarrowia

- Dilute Yarrowia culture 1:000

* Use 1.5 mL microcentrifuge tube

+ Pipette .999 mL of TSB into tube

+ Pipette .001 mL of grown Yarrowia culture into tube

+ Inoculate tube by flicking 4-6 times

- Pipette .300 mL of diluted Yarrowia culture on TSA plate

* Use L-spreader to spread diluted Yarrowia culture

* Let plates dry in fume hood for 30 minutes

- Perform steps 4-6 a total of 3 times on 3 separate TSA plates

- Using a pipette, spot 4 0.005 mL spots of chitinase enzyme on plate

* 2 spots diluted to 10 mg/mL

* 2 spots diluted 5 mg/mL

- Using a pipette, spot 6 0.005 mL spots of chitinase enzyme on plate

* 2 spots diluted to 10 mg/mL

* 2 spots diluted 1 mg/mL

* 2 spots diluted 0.1 mg/mL

- Using a pipette, spot 4 0.005 mL spots of chitinase enzyme on plate

* 2 spots diluted to 0.1 mg/mL

* 2 spots diluted 0.01 mg/mL

- Use one plate without Yarrowia as a negative control

- Take one plate with Yarrowia spread and pipette 4 0.005 mL spots of PBS

* Use plate as positive control

- Cover plates with a plastic bag and place all plates into an incubator

* Incubate overnight at 27°Ce

## Chitinase And Cinnamaldehyde Spotting Testing Against Yarrowia

- Pipette 0.300 mL of diluted Yarrowia culture on TSA plate

* Use L-spreader to spread diluted Yarrowia culture

* Let plates dry in fume hood for 30 minutes

- Make discs

* Using a holepunch, create 100 discs

* Wrap discs in aluminum foil

* Autoclave discs

- Soak 25 discs in each of the following

* Chitinase enzyme from Streptomyces Griseus diluted to 10 mg/mL in 1/32 diluted cinnamaldehyde

* Chitinase enzyme from Streptomyces Griseus diluted to 5 mg/mL with 1/32 diluted cinnamaldehyde

* Chitinase enzyme from Streptomyces Griseus diluted to 10 mg/mL with 1/32 diluted cinnamaldehyde

* Chitinase enzyme from Streptomyces Griseus diluted to 5 mg/mL with 1/64 diluted cinnamaldehyde

- Place 4 of each of the previous discs on Yarrowia covered TSA plates

- Cover plates with a plastic bag and place plates in incubator

* Incubate overnight at 27°C

![](https://static.igem.org/mediawiki/2018/6/6a/T--US_AFRL_CarrollHS--_NavyWhiteDNA.png)

# Biofilm Assay Testing With Cinnamaldehyde

## Growing a Biofilm

- Grow a culture of the wild-type Nissle overnight in a rich medium (i.e. LB)

- Dilute the overnight culture 1:100 into fresh medium for biofilm assays.

- Add 100 μL of the dilution per well in a 96 well dish.

- Add cinnamaldehyde at different volumes to create the desired concentrations

- Incubate the microtiter plate for 4-24 hrs at 37°C.

## Staining the Biofilm

- After incubation, dump out cells by turning the plate over and shaking out the liquid.

- Gently submerge the plate in a small tub of water (i.e., use the bottoms of pipette tip boxes for P1000 pipetman as the tub). Shake out water. Repeat this process a two more times. This step helps remove unattached cells and media components that can be stained in the next step, and significantly lowers background staining.

- Add 125 μL of a 0.1% solution of crystal violet in water to each well of the microtiter plate. Wear gloves and a lab coat while making the solution. Use caution when weighing out the CV as the powder is hydroscopic and readily stains clothing, skin, etc.

- Incubate the microtiter plate at room temperature for 10-15 min.

- Rinse the plate 3-4 times with water by submerging in a tub of water as outlined above, shake out and blot vigorously on a stack of paper towels to rid the plate of all excess cells and dye.

- Turn the microtiter plate upside down and dry for a few hours or overnight.

- For qualitative assays, the wells can be photographed when dry.

## Quantifying the Biofilm

- Add 125 μL of 30% acetic acid in water to each well of the microtiter plate to solubilize the CV.

- Incubate the microtiter plate at room temperature for 10-15 min.

- Transfer 125 μL of the solubilized CV to a new flat bottomed microtiter dish.

- Quantify absorbance in a plate reader at 550 nm using 30% acetic acid in water as the blank.

*Procedure is a modified form of NCBI’s Microtiter Dish Biofilm Formation Assay [(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182663/)*

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Contact us @iGEMLabPats or iGEM@carrollhs.org

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# Team:US AFRL CarrollHS/Results

[![](https://static.igem.org/mediawiki/2018/4/4f/T--US_AFRL_CarrollHS--_WhiteDNALinearLogo.png)](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS)

* Team

  [Team Members](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Team)
  [Collaborations](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Description)
  [Design](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Design)
  [Experiments](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Experiments)
  [Notebook](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Notebook)
  [Interlab](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/InterLab)
  [Model](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
  [Results](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Results)
  [Demonstrate](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Demonstrate)
  [Improve](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Improve)
  [Attributions](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Attributions)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Parts)
  [Composite Parts](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Composite_Part)
  [Part Collection](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Part_Collection)
* [Safety(current)](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Safety)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Public_Engagement)
* Awards

  [Applied Design](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Applied_Design)
  [Best New Composite Part](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Composite_Part)
  [Best Part Collection](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Part_Collection)
  [Education & Engagement](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Integrated_Human_Practices)
  [Model](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
* [Judging Form(current)](https://igem.org/2018_Judging_Form?team=US_AFRL_CarrollHS)

![Results](https://static.igem.org/mediawiki/2018/c/ca/T--US_AFRL_CarrollHS--ResultsHeader.jpg)

# Detect and Deliver

##

The LabPats were able to complete their sense and respond module as planned. The goal of the project was to base a plasmid design very similar to last years. The students wanted a simple circuit design capable of delivering the microbe to the C4-HSL and biofilm. They first started with a constitutive promoter, PJ23117, to continually express RhlR. By continuing expressing RhlR, it would allow for the binding of C4-HSL whenever it became present. Afterwards, it would activate the PRhl promoter and start the transcription and translation of our CheZ gene, which would allow the flagellum of the microbe to navigate in a straight line path. When C4-HSL was no longer expressed, CheZ production would stop and the degradation tag would kick in, allowing the cell to start tumbling again. Hopefully through expressing CheZ and tumbling, a majority of the bacterium will be able to make it to the biofilm.

![](https://static.igem.org/mediawiki/2018/8/81/T--US_AFRL_CarrollHS--RhlRPlasmid.png)

Figure 1. Genetic Circuit for the detect and deliver mechanism

With this goal in mind, the LabPats set to work with the “Detect” and “Deliver” mechanism. In their lab, Dr. Goodson had a similar construct readily available, however the gene expressed was GFP. In the first couple weeks, the LabPats were able to ligate Dr. Goodson’s construct within the iGEM backbone. This plasmid was sent off to IDT and sequenced, and the LabPats were ecstatic to see a perfectly expected sequence. They then utilized primers to insert the CheZ in place of the GFP. This proved to be quite a task as we experimented different methods to insert the CheZ within the plasmid correctly. After multiple failed attempts, the LabPats tried it over and over again, going as far as experimenting with a gradient PCR and ordering brand new primers. Eventually, after a lot of trials, the LabPats were able to send off the final construct to IDT for sequencing, achieving favorable results.

While in the design phase of constructing their plasmid, the students also completed motility tests with *E. coli* cells with the CheZ knocked out. *E. coli* by nature have produce CheZ, so by testing with a strain without CheZ (courtesy of Professor Sandy Parkinson), the LabPats were able to make a clear distinction that the *E. coli* without CheZ knocked out were non-motile. Thus, they could insert their finished plasmid within the non-motile *E. coli* and observe movement towards C4-HSL.

We also performed some experiments with GFP to ensure the detect mechanism worked correctly, and these results can be found on our [Demonstrate](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Demonstrate) page.

![](https://static.igem.org/mediawiki/2018/e/e1/T--US_AFRL_CarrollHS--_WhiteNavyDNA.png)

# Destroy

##

Chitinase C-1 was successfully cloned with the ice nucleation protein into the iGEM backbone plasmid for part submission.
Chitinase B4A was successfully cloned with the ice nucleation protein into the iGEM backbone plasmid for part submission.
The three enzymes in order to produce cinnamaldehyde, which was originally in three parts, was assembled into a DNA vector with two more ribosomal binding sites to improve efficiency, but was not able to be successfully cloned into the iGEM backbone plasmid.

We also performed a number of tests to determine the efficacy of chitinase and cinnamaldehyde against fungi and bacteria, and these results can be found on our [Demonstrate](https://2018.igem.org/Team%3AUS_AFRL_CarrollHS/Demonstrate) page

![](https://static.igem.org/mediawiki/2018/7/75/T--US_AFRL_CarrollHS--ChitinPart2.png)

Figure 2. Genetic Circuit for the chitinase mechanism

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Contact us @iGEMLabPats or iGEM@carrollhs.org