Team:US AFRL CarrollHS/Notebook


Week 1: June 1-2

Thursday (06/01/17)

Exciting first day orientation and safety training off-Base for the newly assembled crew. The mentors and students bonded as work started towards a common goal.

Friday (06/02/17)

On the second day, the students began learning and overviewing basic lab techniques. These techniques include conversion factors, serial dilutions, micro pipetting, and creating and pouring plates of LB agar. Under the tender supervision of knowledgable mentors, the students learned to accurately master lab techniques that would prove invaluable once lab work began.

Week 2: June 5-9

Monday (06/05/17) and Tuesday (06/06/17)

The first two days of this week were spent on Wright Patterson Air Force Base (WPAFB). The team members toured the two labs that they would be working in and received safety training in both. The student researchers were very excited to tour the world-class facilities on base. Multiple brainstorming discussions were also held to finalize the project idea. After the week of training, the group of students split up into 2 groups: one to work in the 711th Human Performance Wing, and one to work in the Materials Directorate. The young researchers at the 711th Human Performance Wing were tasked with sensing and response while the researchers at the Materials Directorate were tasked with packaging.

Wednesday (06/07/17)

Sense/Respond: On the first day in the lab the members made LB broth, (formula in protocol tab). Although a simple procedure, this was the first hands-on experience for the students in the lab. They then did a plasma transformation protocol using E.coli JM109 and the plasmid PRhl_GFPa1. The bacteria created was plated onto LB chloramphenicol and left in incubator (37°C), overnight. If there was growth on the plates in the morning there would a greater probability that the E. coli absorbed the plasmid. The tired but excited young researchers went home with fingers crossed.

Packaging: The lab members attended a review meeting for the Materials Lab. This is where people gave mini presentations on how their projects were going and what problems they have run into. The Packaging members and Chia considered the advantages of using different surface proteins in order to package E. coli and decided on curli. The four Packaging students searched the iGEM site for parts (cellulose binding domains, CsgA, etc) was discussed.

Thursday (06/08/17)

The team attended a seminar given by a professor from Northwestern.

Sense/Respond: When they entered the lab on the second day the plates showed growth with distinctions of individual colonies. Mentor Mike Goodson ecstatically let the students know about the significance of the growth and students were relieved that their plates had grown! These colonies were used to complete the PCR protocol. The master mixture, (protocol tab) and LB broth in round bottom falcon tubes were left in the incubator (37°C), overnight. The plates were put in the refrigerator (4°C) to preserve the bacteria for future use. This protocol was used to confirm that E.coli JM109 had absorbed the plasmid PRhl_GFPa1

Packaging: The Packaging team clarified the goals of their project by deciding to attach a cellulose binding domain to the CsgA portion of a curli protein on E. coli bacteria. In the lab, the team worked with producing agarose gel, diluting a buffer, and using these components to run gel electrophoresis on DNA samples. Additionally, the team poured LB agar plates.

Friday (06/09/17)

Sense/Respond: For the last day of the week, the students’ goal was to confirm the E. Coli had absorbed the plasmid and then perform a MiniPrep Kit to take the plasmid out of the bacteria. To accomplish this, they first learned how to create a gel and how to use the electrophoresis. The electrophoresis sends the DNA down the gel using a positive and negative charge. Under a blacklight you can see if the DNA is present. Next, the students use a QIA prep spin Miniprep Kit to take plasmid out of bacteria. The students placed one nanodrop onto spectrophotometer to measure how pure the DNA extracted is. For future experiments, the plasmid DNA would then be sent to an offsite lab to be sequenced.

260 280 Ratio 260/280 ng/μl
Tina 1.099 0.620 1.77 54.9
Hayley 1.594 0.867 1.84 79.7
Annie 1.366 0.776 1.76 98.3
Jason 1.599 0.886 1.81 79.9

Table 1: Nanodrop results

Packaging: The Packaging team prepared DNA samples and analyzed the samples with the Nanodrop device. Later, the team used restriction enzymes to slice open the DNA and then ran the samples through gel electrophoresis. The project idea continued to progress with the team finding parts form the iGEM inventory and researching DNA sequences of the various parts.

Week 3: June 12-16

Monday (06/12/17)

The whole team met in the morning to discuss the Wiki and each group’s project.

Sense/Respond: The students went to a group meeting within their labs. Students streaked bacteria onto agar plates and put back into the incubator. The rest of the day was spent collaborating on website design and graphic design. Two coding novices, Annie and Jason, worked on learning html, CSS, and Javascript. Graphic designer Tina created a new mascot microbe. Assistant graphic designer and artist Annie created a drawing of the original Wright Flyer and assisted in Photoshop drawing. Stenographer Hayley typed up protocols and helped organize team documents.

Packaging: The RX team observed the preparation and running of PCR. The team made LB broth and agar. Later the team observed the purification of the PCR results and the analyzation of the results with Qubit.

Tuesday (06/13/17)

Sense/Respond: The students removed the agar plates with bacteria from the incubator. Two tubes were filled with LB broth, one labeled as a blank and one containing the bacteria. Using a sterile toothpick, a single colony was picked from each plate, and placed in the culture tube. An unused sterile toothpick was added to the blank tube, to verify that the process was sterile. These tubes were then shaken in an incubator for 3 hours.

The students conducted an experiment using culture growth to see if the bacteria will turn green with the addition of the quorum sensing molecule to the bacteria.

The next morning, the plasmid should glow green indicating it is present.

Packaging: The RX team attended a Materials Lab meeting. Afterwards, the team poured agar plates and transferred LB broth from one liter bottles to Falcon tubes. The team went to the RH lab to obtain the double CBD (cellulose binding domain) DNA from the iGEM plates and transfer the DNA to RX.

Wednesday (06/14/17)

Sense/Respond: The students retrieved the test tubes placed in the incubator the day before and analytically checked the optical density and the fluorescence of the colonies. Immediately afterwards, a visual check was performed as well, confirming that the Colony + 3OC12 glowed green. The ecstatic students celebrated as anticipated results showed through the light.

Colony+3OC12 Colony+DMSO LB+3OC12 LB+DMSO
Dallas 0.929 1.048 0.039 0.037
Tina 1.075 1.084 0.048 524.53
Hayley 1.094 1.079 0.042 0.041
Annie 1.096 1.079 0.044 1.227
Jason 1.229 1.213 0.055 1.227

Table 2: Optical Density of PLCD-GFPa1

Colony+3OC12 Colony+DMSO LB+3OC12 LB+DMSO
Dallas 2359.7 889.14 594.84 645.10
Tina 35454 841.14 524.53 1074.5
Hayley 27733 844.70 508.87 513.41
Annie 28791 837.42 906.98 950.71
Jason 35088 958.79 673.31 950.71

Table 3: Fluorescence of LCD-GFPA1+3OC12

In the afternoon, the students were back at work again preparing a mini-prep on the PLCD-GFPa1 (the plate streaked on Monday). The remaining DNA containing the GFP was then placed in the -20o C freezer for storage. Having much more experience the second time doing the mini-prep, the students had a much better grasp and spent less time with more accuracy.

260 280 Ratio 260/280 ng/μl
Dallas 2.106 1.273 1.65 105.3
Tina 0.927 0.456 2.03 46.4
Hayley 0.723 0.766 2.18 38.8
Annie 0.705 0.357 1.97 35.2
Jason 0.561 0.276 2.03 28.0
Blank -0.50 -0.048 1.04 -2.5

Table 4: Nanodrop results

Packaging: The RX team performed a transformation in order to induce DH5 alpha competent cells to accept a plasmid containing the DNA for the double cellulose binding domain protein. The plates containing the bacteria were left in an incubator overnight in order to allow colonies to grow.

Thursday (06/15/17)

Sense/Respond: Time to go back to work bright and early. A little after the crack of dawn, the engaged students immediately went to work preparing a digest. The scholars made a 15 uL digest along with a Master Mix. Interactive steps included preparing a mini-prep, mixing with enzyme BSr61-HF, enzyme KpuI-HF, buffer Cutsmart, and water. The remaining solution was then placed in incubation for 2 hours. In addition, students were excited to read and learn about plasmid cloning by restriction enzyme digest and restriction endonuclease. With a desire to learn more, the students trekked back to the lab and met with another qualified scientist to assist in the digest. Once the digest was done, the students made a gel and electrophoresis to analyze the DNA. The electrophoresis sends the DNA down the gel using a positive and negative charge and allows the students to see the amount of base pairs, and in this case also allows the students to see the cut DNA and replacement. However, the young scientists were very disappointed to find out the the digest did not go as expected and DNA did not get replaced as expected. Disappointed but not defeated, the students immediately went back to work and retraced their steps, beginning the process all over again. Things do not always work out in the lab and the students were eager to restart and achieve favorable results.

260 280 Ratio 260/280 ng/μl
Dallas- PLas1s 2.429 1.275 1.91 121.5
Dallas- PLas2 1.363 0.701 1.94 68.2
Tina- PLas1 2.059 1.065 1.93 103.0
Tina- PLas2 1.883 0.990 1.89 94.2
Hayley- PLas1 0.994 0.516 1.93 49.7
Hayley- PLas2 1.691 0.883 1.92 84.6
Annie- PLas1 2.186 1.149 1.90 109.3
Annie- PLas2 1.488 0.796 1.87 74.4
Jason- PLas1 1.3494 0.723 1.93 69.7
Jason- PLas2 2.160 1.104 1.96 108.0
Blank 0.024 0.026 0.91 1.2

Table 5: Nanodrop results

Packaging: The RX team performed a Restriction Enzyme Digest on the pBAD containing CsgA DNA and then purified the cut pBAD using a clean-up kit. The team then analyzed the results of the clean-up using the Nanodrop. When the Nanodrop confirmed the presence of DNA, the team created a Master Mix and ran PCR on the CsgA DNA. Upon the completion of the PCR, the products were purified using a purification protocol.

Friday (06/16/17)

Sense/Respond: Immediately the next day, the eager students were right back at work. The previous day's failure had fired them up and they were eager to do everything carefully and quickly. The day began with a student-led mini-prep in which the students were able to follow the protocol and do everything by themselves. Afterwards, the students and mentors went out for a cool ice cream treat. All 12 days of hard work deserved a celebration and the students and mentors bonded over frozen custard and ice cream.

Packaging: The RX team prepared agarose gel and 1X TAE Buffer in order to run the CsgA DNA PCR products on a gel. Images were taken of the results of the gel electrophoresis in order to confirm the presence of the correct DNA. Colonies began to appear from the transformation using the dCBD DNA.

Week 4: June 19-23

Monday (06/19/17)

Sense/Respond: After the weekend, the students met in the lab and initiated a second digest. They were determined to get everything measured correctly and leave minimal room for mistakes. This time around, the students were much more knowledgeable about buffers and enzymes used and proceeded carefully combining them with DNA. After the digest was complete, the young scientists began prepping another gel for electrophoresis. Everything went smoothly while the digest was completed and DNA was extracted and cut by the enzymes. Next, the students prepped the DNA by adding a loading dye and then proceeded to put it on the gel. After electrophoresis for 30 min, the gel was complete. The LabPats were able to use this downtime for updating lab journals, creating new and exciting logos, as well as continuing to work on the wiki. Next, another experienced mentor helped with analyzing the gel and cutting out the DNA from the gel. However, they were quite disappointed to learn that there were 3 bars, indicating more base pairs than expected. The mentors ensured them that the gel still worked and that they just needed to cut out the fluorescent band that was relevant. Meticulous work was done to cut out the gel, and the labpats were finally able to let out a sigh of relief once the gel was safely placed in the tube and put in the fridge to be melted down tomorrow.

Packaging: To start the week, the RX team ran a miniprep and restriction digest on the dCBD DNA. After the restriction digest, the team ran a gel to test the different digested DNA lengths. The gel results weren’t as expected and the team began to re-do a miniprep.

Tuesday (06/20/17)

Sense/Respond: The students quickly resumed where they had left off to begin their gel extraction. Two different protocols were used to see which one would achieve a higher concentration. The teams were divided into Tina and Annie working with Svetlana on (QIAEX II Agarose Gel Extraction) and Jason and Dallas (who unfortunately was ill, so Annie and Tina tag teamed his sample) working with Mike on QIAEX Gel Extraction Kit. The gel extractions were performed and the nano drop test was performed. The only successful gel extractions were performed by the girls (Svetlana’s protocol), so they took home the victory! The boys learned a lot from their error and submitted defeat. The day was wrapped up by working hard designing the wiki and learning how to code.

Packaging: The RX team finished the miniprep from Monday and ran another nanodrop test to make sure of the concentration and purity. Later, the team prepared LB broth with Kanamycin. To increase the amount of DNA, the RXers ran a PCR and then prepared a gel to test the PCR products.

Wednesday (06/21/17)

Sense/Respond: Today the students began their second Polymerase Chain Reaction (PCR). Under the watchful eye of Dr. Harbaugh, the students independently finished the PCR and loaded it into the thermocycler. Another small gel was prepared and the PCR’s were taken out and loaded on the gel. The students meticulously loaded the PCR’s onto the slits of the gel with incredible precision that truly showed their growing expertise.

The students received the results they had been hoping for, which showed that the Super folded GFP had been inserted into the bacteria. They then performed a PCR Purification, which separated the massive amounts of DNA. The DNA was then measured using the nano drop and these results were given:

260 280 Ratio 260/280 ng/μl
Dallas 3.099 1.700 1.82 155.0
Tina 2.355 1.357 1.74 117.8
Jason 1.756 1.005 1.75 87.8
Hayley 2.3 1.3 1.81 119.6
Annie 3.793 2.050 1.85 189.6
Blank 0.013 0.058 0.022 -0.6

Table 6: Nanodrop results

Packaging: The RX team ran another gel using the PCR products of dCBD DNA from Tuesday. After the gel was complete, the DNA bands were cut from the gel and purified using a gel extraction. The results of the gel extraction were run on the previous gel in order to confirm the dCBD DNA as correct. The dCBD DNA was injected into a pCR Blunt II TOPO plasmid through a TOPO Ligation and then transformed into TOP 10 competent cells.

Thursday (06/22/17)

Sense/Respond: The day began with another PCR. Today, the students were ready to completely do the PCR by themselves with little guidance from the mentors. The standard protocol was followed and the PCR and ran another cycle in the thermocycler. Meanwhile, another gel was prepared by the students. This time around, the students were very efficient and the process went by very quickly. The gel ended up giving too many bands, but the mentors conversed and decided to proceed with the PCR purification anyways. After the lab work was done, the students decided to meet with the other lab students to discuss current progress. The students created discussion points and had a great discussion with the other lab.

Packaging: The morning began with the RXers beginning cellulose cultures. Later the team started colonies of the TOP 10 E. coli cells in culture tubes with LB broth with Kanamycin, as well as LB agar plates with Kanamycin. Growing colonies would allow the plasmids to replicate inside the cells. The entire team met at UES in the afternoon.

Friday (06/23/17)

Sense/Respond: The day began bright and early with two PCR digests. This time a PCR Vector Digest was conducted on the same from yesterday and a PCR GFP digest was done on the sample from Wednesday (6/21/17). After following the protocol, the students were able to successfully perform the digests. After a 2 hour wait (per the protocol), the young scientists performed a purification on the GFP digest. The goal was to cut off end bits of the base pairs to allow the plasmid connect again. During the students' lunch break, another meeting was held with teacher leaders who wanted to know information about progress and future proceedings.

260 280 Ratio 260/280 ng/μl
Dallas 2.106 1.273 1.65 105.3
Tina 0.927 0.456 2.03 46.4
Jason 1.188 0.719 1.65 59.6

Table 7: Nanodrop results

DNA 10μl
10x Buffer 1.5μl
Kpn1-HF 1μl
BsrG1-HF 1μl
H2O 1.5μl

Table 8: GFP Digest (15μl)

DNA 8.5μl
10x Buffer 1.5μl
Kpn1-HF 1μl
BsrG1-HF 1μl
H2O 3μl

Table 9: Vector Digest (15μl)

Packaging: The RXers extracted the TOPO plasmids from the E. coli and ran a restriction digest. To ensure the correct dCBD length, both cut and uncut DNA was run on a gel. The results of the gel determined the possibility of sending in plasmids with dCBD for sequencing on Monday.

Week 5: June 26-30

Monday (06/26/17)

Sense/Respond: The students began another week of hard work. They made a gel to run their previous Plasmid Digest. Next, they looked at the gel under the blue light and found the band they were looking for at 3.2k base pairs. Next they scalpeled out the desired band and began a gel extraction using the QIAEX ll Agarose Gel Extraction Protocol. After the gel extraction they tested their results on the Spectrotropsesis. The first sample was lower than preferred and the next sample had a very uneven graph, but with the desired numerals. Afterwards, the mentors discussed and determined to still do a legation the next day and see the results.

260 280 Ratio 260/280 ng/μl
TD+AB+JD 0.046 0.027 1.71 2.3
DM+HJ 0.227 0.183 1.24 11.3
Blank 0.015 -0.005 -2.86 0.7

Table 10: Nanodrop results

Packaging: The RX side of the team did a nanodrop on the uncut dCBD TOPO plasmid and found the concentration of each sample. Water was added to each samples to create an equal concentration at 50 nanograms per microliter and 500 ng in all. We then sent our samples to be sequenced.

Tuesday (06/27/17)

Sense/Respond: Today was the student's first ligation. The goal was to put the sticky ends back together to form a complete plasmid. A lot of math was done to determine the exact ratios of sfGFP and PCR vector plasmid. Luckily, Dr. Harbaugh was able to assist the students with newly introduced lab techniques. There was another protocol and the students were able to follow it with no problem. They then went to the conference room and had another meeting with the other labs and teachers. This time, both labs gave extraordinary presentations about project progress and end goal. The RH lab presented their abundance of lab work and myriad of website/wiki work. The RX lab presented lab work and some technical website work. Afterwards, the groups planned out agenda and discussed necessary steps to complete the wiki page.

Packaging: The team removed the cellulose from the plates, rinsed the cellulose top layer and put each sample in a test tube. The team took pictures of each sample. The tubes were then frozen in a -80༠ freezer.

Wednesday (06/28/17)

Sense/Respond: The students began the day with a new PCR. After examining the plates from yesterday, it was determined that there indeed was green fluorescence on the plates. However, a PCR was needed with a gel to determine whether or not it was sfGFP or GFP. High in spirits, the students immediately went to work. First, they created a Master Mix following the PCR Protocol for Phusion DNA Polymerase. They did some tricky math and figured out exact proportions needed for nuclease-free water, Phusion Buffer, DMSO, Kpn1(forward primer), BsrG1(reverse primer), as well as the phusion DNA polymerase. They each used 6 colonies. Four students used green colonies and then dipped it into the PCR and the LB Broth with chloramphenicol. This method ensures that only the bacteria immune to chloramphenicol can survive. On the gel, it should give a precise band of base pairs indicating sGFP or GFP. Another student used 6 colonies without the green fluorescence. It acts as a control. When it is run as a gel, there should be random bars that indicate there is no sGFP or GFP. Then the samples were put into the thermocycler for 1.5 hours according to the protocol. The students then had the endure a nerve raking wait as they wondered if the PCR had gone successfully and whether the bacteria had sGFP or not. After preparing another gel, the students meticulously put the samples on the 40 wells. After 30 minutes of running the gel, the students came back to an ecstatic Dr. Goodson informing them that the gel had turned out as planned and that they could proceed with the mini-prep before sending in the DNA for sequencing. Everyone shared "elbow-fives" and were relieved that all their hard work had not gone to waste!

Packaging: The cellulose tubes were removed from the -80༠ freezer. The tubes were then put into a beaker that would be attached to a lyophilizer overnight at -105༠ in a vacuum.

Thursday (06/29/17)

Sense/Respond: Today the students began to mini-prep the samples from the previous day. Right from the start, the students noticed fluorescent green coming from one of the tubes. This would foreshadow what they would see later in the day. They began the mini-prep full of hope and concentrated on following the protocol exactly to achieve the best possible results. They decided to first put the tubes over the light and observed a lot of fluorescence (see picture). After about 2 hours, they were finally done. They nano dropped the sample and observed an incredible amount of DNA. Everyone cheered as they realized that the "response" component of the project was successful.

Packaging: Sequencing results for dCBD and CsgA DNA were reviewed using the program Blast. Each of the six dCBD in TOPO plasmid samples contained no errors on either the SP6 or the T7 primer sequences; however, some samples contained the gene in the reverse orientation. Chia's CsgA in TOPO plasmid samples were also entirely correct.

Friday (06/30/17)

Sense/Respond: Since all the lab work is currently done until the DNA sample is sent to Ohio State University for DNA sequencing, the students had the whole day to make website improvements and wiki renovations. New logos were also created as well as graphics. The students will continue to add content to the wiki weekly.

260 280 Ratio 260/280 ng/μl
1 2.899 1.657 1.75 145.0
2 2.364 1.268 1.86 118.2
3 1.948 1.030 1.89 97.4
4 2.152 1.143 1.88 107.6
5 2.071 1.123 1.84 103.5
6 2.421 1.318 1.84 121.1
7 2.072 1.103 1.88 103.6
8 1.764 0.963 1.83 88.3
9 2.287 1.228 1.86 114.4
11 6.402 3.492 1.83 320.1
Blank 0.0003 0.029 0.11 0.2

Table 11: Nanodrop results

Packaging: The RXers learned how to design a restriction digest and created the digests for CsgA, dCBD, and pBAD samples. Later, the team performed the three digests, each with one sample, and left the samples to incubate in a heat block for several hours. In the afternoon, the whole iGEM team met with mentors to review the projects.

Week 6: July 5-6

Wednesday (07/05/17)

“Ring, ring, ring!” came from the laptop as the students huddled around to receive a Skype call from the Singapore iGem team. They were greeted by Wilbert from the Singapore team. They immediately began conversing with Wilbert and exchanged project descriptions and ideas. The team was very intrigued to learn about the Human Practices components of iGem from Wilbert. After a whole hour of video conference, the team learned a lot from the sagacious Wilbert. The conversation was very beneficial to the students as they learned about the different components of iGem. Wilbert even offered to help them with the wiki page!

Sense/Respond: Afterwards, the students arrived back to the lab and received presentation tips from mentors for the upcoming lab presentations the following week.

Packaging: The entire iGEM team convened to hold a Skype call with a representative from the Singapore National College's team. Later on Base, the RXers ran a gel on the results from the previous Friday's digests, which included pBAD, pCRBlunt-CsgA, and pCRBlunt-dCBD. Upon completion, the team members extracted DNA for each sample from the gel using a gel extraction protocol.

Thursday (07/06/16)

Sense/Respond: The next day, the group decided to do the InterLab in order to be considered for medal criteria. They sifted through the kit and found many useful parts that they could use in order to help standardize fluorescence. After reading through the protocols and laying out a plan of attack, the students had the wait since the lab was going under routine inspection. The students spent the rest of the day designing more graphics and made wiki edits.

Packaging: The morning began with running a gel on the results of the gel extraction. While the gel was solidifying, the team determined the concentrations of the samples with the Nanodrop. After the gel confirmed the pBAD, csgA fragment, and dCBD fragment, the RXers performed a ligation in order to clone the csgA gene into the pBAD plasmid.

Gel Extraction Results Concentration (ng/uL) 260/280 260/230
pBAD 1 (w/ Ncol and Kpnl) 6.2 1.64 0.02
pBAD 2 (w/ Ncol and Kpnl) 7.4 2.11 0.01
CsgA 1 (w/ Ncol and Kpnl) 17.0 1.88 0.03
CsgA 2 (w/ Ncol and Kpnl) 21.5 1.79 0.05
dCBD 1 (w/ Ncol and HindIII) 8.0 1.75 0.02
dCBD 2 (w/ Ncol and HindIII) 8.8 1.98 0.02

Table 12: Nanodrop results

In the afternoon, the whole team got together with mentors to meet at the Air Force Museum in Dayton, Ohio in order to take self pictures and group pictures for the wiki.

Friday (07/07/16)

Packaging: To finish the week, the RXers transformed pBAD-CsgA plasmids into DH5-alpha competent cells and patched the samples onto LB agar plates with ampicillin. Additionally, the team made and poured a multitude of LB agar plates with ampicillin. The entire team met at UES in the afternoon to discuss the Wiki.

Week 7: July 10-14

Monday (07/10/17)

Sense/Respond: At 9 AM, the students went to their weekly lab meeting. Interns from their department always have a weekly meeting where they share ideas and summer progress. The LabPats will share their presentation next week. Afterwards, it was finally time to begin the InterLab study for the iGem project. The students began re suspending the wells. However, they soon found out that there were no more plates of LB- Chloramphenicol and thus had to make more. They had to postpone the InterLab studies and finished the day by creating a lot more plates for future experiments.

Packaging: To start the week, the RX team made LB Broth with Ampicillin and later transferred colonies of E. coli cells with the pBAD-CsgA plasmids to allow them to grow. To accomplish this, the RXers patched colonies of the cells onto LB agar plates with Ampicillin and then transferred the remainder of the colonies into culture tubes containing LB Broth with Ampicillin.

Tuesday (07/11/17)

Sense/Respond: The student researchers began the InterLab the next morning. They split up in 2 groups while one group worked with the competent cells kit and completed Day 1. The other group transformed the wells inside the plates provided by iGem. The students did a great job following protocol and growing the bacteria on the agar plates from the previous day. After they were done, the students began filling out the necessary forms for the InterLab as well as planned out the dropdown menu and the slideshow for the website.

Packaging: The RXers spent the morning miniprepping twenty-one samples of DH5 alpha competent cells with pBAD-CsgA. Around five milliliters of each sample from the culture tubes of the previous day were pelleted for miniprepping and one milliliter was reserved for storage.

Wednesday (07/12/17)

Sense/Respond: The next morning, the young students attended a lecture taught by biologists from the Purdue University. It was very interesting and higher level, and the students were very excited to hear topics relevant to their iGem goals. Afterwards, they returned to the lab with their mentor to examine the overnight bacteria colonies. However, they were disappointed to learn that the competent cell test did not work out. Instead of an expected 300+ colonies, the plates only yielded about 25 colonies. The students spent the rest of the day redoing the protocol in hopes of achieving the needed amount of colonies in order to proceed.

Packaging: The Nanodrop was used to check the concentration of several samples of the miniprep results. Then, a restriction digest was performed on the miniprepped pBAD-CsgA plasmids with NcoI and KpnI. Digested and undigested pBAD-CsgA samples were run on a gel in order to confirm the lengths of the fragments. The samples with the clearest CsgA bands were chosen to have a second restriction digest with KpnI and HindIII performed on them to prepare for cloning the dCBD gene into the plasmid.

Spot Samples of pBAD-CsgA Concentration (ng/uL) 260/280 260/230
AS2 pBAD-CsgA 100.0 1.90 1.91
AS6 pBAD-CsgA 105.2 1.89 1.93
AP1 pBAD-CsgA 102.1 1.94 2.00
AP2 pBAD-CsgA 106.0 1.88 1.71
MH1 pBAD-CsgA 109.7 1.94 1.92
MH7 pBAD CsgA 64.8 1.92 2.23

Table 13: Nanodrop results

Chosen Samples of pBAD-CsgA Concentration (ng/uL) 260/280 260/230
AS3 pBAD-CsgA 129.2 1.96 1.98
MH2 pBAD-CsgA 83.1 1.94 1.89
MH3 pBAD-CsgA 114.3 1.96 1.98
MH5 pBAD-CsgA 93.4 1.94 1.97
MH6 pBAD-CsgA 108.0 1.96 2.06

Table 14: Nanodrop results

Thursday (07/13/17)

Sense/Respond: Continuing the next morning, the students immediately continued the Interlab. Today they focused on completing the OD600 and Serial Dilution part of the protocol. The students hope to test the fluorescence of the colonies tomorrow. Afterwards, they picked colonies and left them in the incubator to grow overnight. Afterwards, they got back on wifi and successfully implemented a background to the wiki. Tomorrow they will have s video conference with the U.S. Navy and U.S. Army iGem team.

Packaging: The morning began with a "gel extraction" of the digest with pBAD-CsgA with KpnI and HindIII in order to purify the samples. Later, a gel was run to confirm the digested samples of pBAD-CsgA and several samples of dCBD. A number of cultures containing Top10/pCRB-dCBD and separate cultures wtih Top10/pCRB-CsgA were frozen down in a -80 degree Celsius freezer. The day ended with a ligation of the dCBD gene fragment into the pBAD-CsgA.

Friday (07/14/17)

-“I brought doughnuts!” exclaimed Andrea as she walked in the door in the morning. The group had planned a Skype meeting with the U.S. Navy and U.S. Army. After getting the connection set up, the students were able to have a myriad of conversations from topics regarding to giant Jamboree to technical components of the wiki. After some casual conversion, the students were able to have a better understanding of the happenings at the Jamboree and how to improve the wiki as well as Interlab components.

Sense/Respond: Next, the long part of the Interlab began. The students tested for absorbance and fluorescence of the bacteria samples over 0,2,4, and 6 hours. Later in the afternoon, the students did a dry run of their presentation in preparation for Monday.

Packaging: The entire iGEM team participated in a video conference with the Army and Navy iGEM teams in order to discuss similarities and issues with managing military teams. Afterwards at the lab, the RX team transformed the ligation into NEB 5-alpha competent cells and left the cells to grow in an incubator over the weekend.

Week 8: June 17-21

Monday (07/17/17)

Sense/Respond: The student sat around nervously waiting for their presentation to begin. In a few minutes they would be presenting to the whole lab about their project and goals. The students gave a phenomenal presentation that received much praise and applause. They were relieved to be done and went back to work on the wiki. Everything in the lab is up to date so the young researchers dedicated the rest of the day for wiki organization and miscellaneous tasks.

Packaging: GoTaq Flexi PCR protocol was performed on 28 colonies of the transformants. After the PCR was completed, a gel was run in order to verify which of the chosen colonies contained both the csgA and the dCBD gene fragments. Based off of the PCR products, 6 of the 28 chosen colonies were grown on LB with ampicillin plates and broth in an incubator overnight.

Tuesday (07/18/17)

Sense/Respond: Mentor Mike was finally back from vacation and the students were very excited to see him and update him on current progress. They discussed the plan moving forward and the layout of the rest of the summer. After half an hour, the students had a general idea of the "end goal". They discussed the DNA sequencing that had just come in and were like described. Afterwards the students continued working on collaboration and organizing wiki pages.

Packaging: The six liquid culture samples were taken out of the incubator and 3ml from each tube was miniprepped for four restriction digests. The Nanodrop was used to check the concentration of DNA in each sample. After the digests were performed using Nco1, Kpn1, HindII, and Nco1 and HindIII, a gel was run to analyze the results. The results obtained were unexpected, but several samples, 14 and 32, were chosen as likely candidates.

Chosen Samples of Prepped DNA Concentration (ng/uL) 260/280 260/230
Sample 14 8.3 1.61 1.50
Sample 15 33.0 1.49 0.71
Sample 110 21.0 1.67 0.82
Sample 22 17.6 1.69 1.30
Sample 32 43.6 1.75 1.85
Sample 34 23.7 1.06 1.59

Table 15: Nanodrop results

Wednesday (07/19/17)

Packaging: In the morning, the entire iGEM team met with mentors at RH to discuss the projects and Wiki. At the lab, the RXers miniprepped samples 14 and 32 of NEB 5-alpha/ pJAAM1. Later, restriction digests with Nco1, Kpn1, HindII, and Nco1 and HindII were performed on the miniprepped samples. Lastly, cultures of NEB 5-alpha/pJAAM 1 were diluted and spread on LB agar with ampicillin plates.

Thursday (07/20/17)

Sense/Respond: The day started at UES and the students began to look up literature that would help them with the sensing part of the project goal. They were able to find lots of useful information referring to the two ETEC toxins, a heat labile and heat stabile. Over the course of a couple hours, the students were able to find and compose a list of the possible ways to detect ETEC. In the afternoon, the students shared the ideas with their mentor, Mike. After lunch, the students discussed what they had gathered from the past 3 days of researching literature. After exchanging ideas and articles, the students and Dr. Goodson realized that being able to sense a quorum sensing molecule autoinducer-2 (AI-2) might be the way to go. They discussed at length the necessary components needed to be able to put the promoter into the existing plasmid so that it could detect AI-2. After a bit of research, they decided to go with E. coli DH5a which fit perfectly as it does not create AI-2 on its own. The students were relieved that they had taken a massive step forward in their “sensing” component and hope to achieve the end goal soon.

Packaging: A gel was run on the digested pJAAM1 and the results were again unexpected. To try a different approach, the RXers chose colonies from the diluted cultures of NEB 5-alpha/pJAAM1 and performed a PCR. The gel of the PCR products appeared correct, so the remainder of the chosen colonies were streaked onto plates and inoculated in culture tubes to grow overnight.

Friday (07/21/17)

Sense/Respond: The students finally began the lab work today for "sensing". The students worked on resuspending part K18 in Well 3 and followed the protocol to grow them up on plates. They also transformed the GFPa1 plasmid into DH5a E. coli cells. The plates were then left outside overnight to grow. Meanwhile, the young researchers were able to analyze the plasmid and look at all the restriction sites to find the ideal sites to cut the plasmid. Under the guidance of Dr. Goodson, the students picked three restriction sites and an order was put in to order the primers. Afterwards, the RH lab held their weekly lab meeting with the RX lab and shared the exciting news of their new discovery.

Packaging: The week ended with the RX team miniprepping three milliliters of the cultures from the previous day and digesting the results. However, when a gel was run on the digest, the results still appeared incorrect. Consequently, the RXers decided to return to a previous step in order to retry the ligation between pBAD-CsgA and dCBD. Previously digested samples of pBAD-CsgA with KpnI and HindIII were further cut with the same enzymes to achieve a more thorough digestion.

Week 9: July 24-28

Monday (07/24/17)

Sense/Respond: The day was a bit more relaxed for the Monday morning. The weekly lab meeting was scheduled and the students were able to learn a lot about other researchers and their current studies. In addition, they learned how to present to an audience and create simple but informational slides. Afterwards, they enjoyed a team lunch together with mentors to discuss the Michigan meet up. The ecstatic students exchanged travel ideas and a powerpoint presentation for the "mini- jamboree". Afterwards, the young researchers returned to the lab to pick colonies from Friday to grow overnight in LB-Chl. They then left for the afternoon to get on wi-fi and worked on the wiki and human outreach goals.

Packaging: Lab Fun Day!

Tuesday (07/25/17)

Sense/Respond: The adventures continue the next morning as the team went back into the lab to look at the overnight cultures. Tina and Hayley were extremely pleased to find their cultures had grown perfectly and were ready to do the mini-prep and nano drop. However, Dallas and Jason did not get favorable results. They were very disappointed to find out that their cultures had not grown and would need to restart and pick other colonies.

Packaging: The RXers attended a branch meeting and then worked to develop presentations for the Journal Talk on Friday, and for Michigan State. Later, the team performed a gel extraction protocol on the digests from Friday and created a gel for gel electrophoresis on Wednesday.

Wednesday (07/25/17)

Packaging: The entire iGEM team met in the morning at RH to practice the Michigan State presentation and to discuss the trip. Later the RXers ran a gel of the gel extractions, as well as checking the concentrations with the Nanodrop. The results were not entirely desirable, likely due to the number of protocols performed with the samples, but a ligation was set up and performed. Vanessa helped the team gather pictures and begin work on their Friday presentation.

Thursday (07/27/17)

Another meeting was held to discuss the presentation and viability of the Michigan State Trip. Students from both labs went over their combined presentation and rehearsed thoroughly.

Sense/Respond: Hayley and Tina prepared cultures of GFPa1 and sfGFP from the plates. Dallas transformed the iGem cultures but sadly failed. Dr. Goodson recommended that they regrow the plates and proceed again tomorrow. Later in the day, the young researchers finalized their plan for "flat Stanley" and the outreach survey they hope to do in the future.

Packaging: To start the morning, the RXers spent a portion of time discussing the Friday presentation with Vanessa. Later the team performed a transformation of the ligation from the previous day and continued preparing for their presentation.

Friday (07/28/17)

The morning began bright and early with a tour of a C-17 cargo plane on base. It was a great bonding experience for teams from both labs as they took pictures and learned about the C-17 aircraft. The RX group mentally prepared to present their project later in the afternoon to some high-ups and members of their lab. The RH Lab planned to attend and give them support. The presentation went very well and the RX lab members received a stellar ovation.

Sense/Respond: Later in the afternoon, the RH lab would be preparing a mini-prep for the Well 3, 18K competent cells provided by iGem. The students had just found out yesterday that the cultures finally worked and that they could start proceeding to the next step, which was extremely exciting. As experts in the lab now, the group was able to follow the protocol with ease and completed the protocol accurately and quickly.

Packaging: Both the RH and the RX teams participated in a tour of a C-17. Afterwards, the RXers presented their project at a Journal Talk. Vanessa suggested troubleshooting by sending samples of pBAD-CsgA off for sequencing. Additionally, the University of of Michigan mutant strains of E. coli arrived. Once the preparation for sequencing was completed, the team met at UES for a bit before leaving for the weekend (Michigan State!)

Week 10: July 31-August 4

Monday (07/31/17)

Sense/Respond: The morning started with the students performing a digest over the mini prep from several days pervious. Two digests were preformed using different sets of cut enzymes. Digest 1 was using the SpnI-HF and BmrI, while Digest 2 used the cut enzymes PacI and BmrI. The Digest was then confirmed using a gel, which showed the accurate number of base pairs.

Packaging: The week started with the RXers streaking and growing cultures of certain colonies of the transformants from Friday. The samples of pBAD-CsgA were sent off for sequencing and a plan was drawn up to further troubleshoot and proceed with the project.

Tuesday (08/01/17)

Sense/Respond: The students set up another run of their GFP Project. The students used a Kinetics reading of the Plate Reader. The kinetics were taken over 24 hours, every 20 minutes. The students then worked on the wiki, trying to create a template.

Packaging: The day began with developing a plan for troubleshooting the project throughout the week. Later, the RXers made up a stock of LB agar and LB agar with Kanamycin and miniprepped three milliliters of the liquid cultures from Monday. Restriction digests were performed on each miniprep and a gel was run, and one sample's results seemed hopeful. New restriction digests were performed on the sample, but the outcomes of the sequencing arrived and suggested that success with the new restriction digests would be unlikely. The sequencing results showed a fragment of the TOPO plasmid had cloned in before csgA in each pBAD-CsgA sample. Solutions of magnesium chloride, calcium chloride, and glycerol were made in order to perform a competent cell protocol on the mutant strains of E. coli, which were left in an incubator overnight to grow.

Wednesday (08/02/17)

Today was an exciting day for the students as they got to perform their survey at their high school. The iGEM students volunteered to help the high school on schedule pick up day. As the other students were coming to grab their schedule we would offer for them to fill out a pick survey on computers we had set up. Over 200 students varying in with some teachers filled the survey. This was a huge success for the students.

Packaging: The RXers chose samples of the pBAD-CsgA containing TOPO plasmid to digest with NcoI in the hopes that the TOPO fragment would separate from the rest. A gel showed the digests to be successful, and a gel extraction was performed. The concentrations of the results from the gel extraction were checked using a Nanodrop. Lastly, the mutant strains with csgA and without csgA were each inoculated onto plates with LB and Kanamycin and with just LB respectively, and these plates were stored in an incubator overnight to grow.

Thursday (08/03/17)

Sense/Respond: Today the students got back to work on the sense plasmid. The students performed a PCR, because finally their primers came in. The PCR product was ran on the gel and the students got the results they were hoping for. Next, they performed a MiniElute Reaction Cleanup Kit and then conducted a Nanodrop. The Nanodrop were high concentrations of DNA. To end the day, the students performed another digest using the PCR they had just purified.

260 280 Ratio ng/ul
F1 7.408 3.974 1.86 370.4
F2 8.412 4.554 1.85 420.6
Blank 0.089 0.002 5.59 0.4

Table 16: Nanodrop results

Packaging: Using the gel extractions from Wednesday, the RXers performed four ligations. After two hours, part of the ligations were removed in order to complete several transformations, while the remaining ligations were left to incubate overnight. Colonies from the plates containing the University of Michigan strains were transferred to flasks containing LB Broth and LB Broth with Kanamycin.

Friday (08/04/17)

The big day to present to the Chief Scientist of the lab had finally come. Early in the day, the students had a dry presentation with their teachers and mentors. They received criticism and praise for their work and presentation that they would present later. They left in high spirits to return to the lab and begin the purification, ligation, and transformation of the plasmid for the “sense” component of the project.

Sense/Respond: The purification was then nanodroped and the students were pleased with the results. The students then performed a ligation on the plasmid backbone from the iGEM part and the sfGFP. The ligation seemed to go as planned and the students hoped that the two sticky ends had connected. After completing the transformation, they got ready to present to the Chief Scientist who got the funding for the iGem team. The presentation went extremely well and the Chief Scientist was very impressed with their work in the summer and the progress made. The students were finally able to let out a sigh of relief as they finished their presentation successfully. Afterwards, they came back to the lab to perform the transformation on the plasmid. The students followed the protocol exactly and were able to achieve favorable results.

Packaging: The entire iGEM team met at Carroll in the morning to explain the project to several teachers from the science department and to the principal. Later, the team presented to the chief scientist of the 711th. In the lab, a second transformation was performed using the ligations that were left overnight. Additionally, samples from the flasks containing the University of Michigan strains were prepared for storage in the negative eighty degree freezer.

Week 11: August 7-11

Monday (08/07/17)

Sense/Respond: Unfortunately the students were disheartened to learn that none of the plates grew colonies. The students were at first shocked, but immediately started tracing back to the different protocols and plasmids from the past few days. Mentor Mike ultimately recommended them to run another gel on the PCR product and the students diligently began the gel protocol. After a few hours, the gel inspection seemed fine and the bands displayed exactly what the students expected. Therefore Mentor Mike recommended that they re-do the ligation from Monday. The ligation went very smoothly and the transformation will be done tomorrow.

Packaging: Samples of pJAAM01 from early August were digested using Nco1 in the hope that the TOPO fragment would separate from the pBAD-CsgA-dCBD plasmid. A gel showed the digests to be successful, and a gel extraction was performed. The concentrations of the results from the gel extraction were checked using a Nanodrop. The RXers then set up ligations using the gel extractions and a gel was run to confirm the gel extractions. Colonies from the transformants of pBAD-CsgA from last week were patched on an agar plate and inoculated in culture tubes. Finally, plates were streaked using the freezer stocks of the University of Michigan strains and left to grow overnight.

Tuesday (08/08/17)

Sense/Respond: Today the LabPats were finally able to transform the plasmid from the ligation yesterday. They followed the standard protocol and transformed the cells into E. coli DH5a again. After that, the cells were ready to be plated like previously. This time the students hope that the cells will grow colonies and indicate the completion of their "sense" component. The students left the lab in high spirits and headed out for wifi and collaboration space to discuss more wiki work and future work.

Packaging: We weren't in the lab!

Wednesday (08/09/17)

Sense/Respond: The students eagerly went into the lab to check the plates from the transformation. To the students dismay all the plates contained growth even the blanks. The concerned students went to ask Mentor Mike, who help the student decide to perform a PCR in order to run the product on a gel. They ran the gel and got the desired band lengths for everything. They also noticed that some of the colonies were already glowing green. With already putting in a long day of work the students made overnights for the next day.

Packaging: We weren't in the lab!

Thursday (08/10/17)

Sense/Respond: Bright and early the students got their overnights out of the incubator. They then made glycerol stocks and performed a mini prep, so the bacteria could be sent for sequencing. In the afternoon, the students got to view a set of poster presentations. This was especially helpful for the students, and gave them many ideas on how to set up their poster.

Packaging: We weren't in the lab!

Friday (08/11/17)

Sense/Respond: Today was a sad day in the lab, as the summer was ending and the students had to return to school. The team spent the morning taking their mentors to breakfast, as a thank you for all they had done for us. The rest of the day was spent at the lab. The students cleaned up their work benches, and made sure everything was in order before they left the lab for the last time. The students said their good byes to their coworkers, whom they had enjoyed many lunch breaks with.

Packaging: We weren't in the lab!

Parts Submission


The parts were submitted according to the protocol provided by iGEM. Minipreped DNA was placed into their respective wells before being dried in a hood overnight. The next day, they were then packaged and shipped.