iGEM 2019 — Team US_AFRL_CarrollHS

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# Team:US AFRL CarrollHS/Notebook

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* Team

  [Team](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Team)
  [Collaborations](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Collaborations)
* Project

  [Description](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Description)
  [Design](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Design)
  [Experiments](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Experiments)
  [Notebook](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Notebook)
  [Contribution](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Contribution)
  [Model](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
  [Measurement](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Measurement)
  [Results](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Results)
  [Attributions](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Attributions)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Parts)
  [Basic Parts](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Composite_Part)
* [Safety](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Safety)* Human Practices

    [Human Practices](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Human_Practices)
    [Education & Engagement](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Public_Engagement)
  * Awards

    [Medal Criteria](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Medal)
    [Measurement](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Measurement)
    [Model](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Model)
  * [Judging Form](https://igem.org/2019_Judging_Form?team=US_AFRL_CarrollHS)
  * [Wiki Guide](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/WikiGuide)

Notebook\_Real\_Header\_Final

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# Notebook

# 6/3/2019 - 6/7/2019

## Monday

### Lab 1:

Lab Pats began by listening to safety training and engaging in
a walk-through of lab facilities, including fire extinguishers,
equipment, and safety locations. Wright Scholars in-processed. Lab
Pats created and autoclaved LB Agar and engaged new members in
micro-pipette training. New members also learned to use gel
electrophoresis equipment. Poured plates - chloramphenicol. UES
Members in-processed.

Located RhlR plasmid (glycerol stock #7 in Amy's box). Streaked LB-Chl
plates and placed in incubator at 37°C overnight Began working with
benchling.com program.

### Lab 2:

## Tuesday

### Lab 1:

### Lab 2:

The RX side of the team showed up to lab excited and eager to get back to work from having the school year off. After fully becoming employees at UES Inc., the students headed to lab to start off the year. The students met with mentor Dr. Chia Hung and discussed all of the plasmid designs that would be needed to be completed by the end of the week. Also the students attended safety training and lab protocols in order to ensure a safe work environment. After the training the team met at UES in order to begin work on the plasmid designs.

## Wednesday

### Lab 1:

Located RhlR plasmid (glycerol stock #7 in Amy's box). Streaked LB-Chl plates and placed in incubator at 37 oC overnight Began working with benchling.com program

### Lab 2:

On the second day of returning to work, the RX team met early in the morning in order to discuss the day's plans. They met with Dr. Vanessa Varljay in order to once again discuss and design the plasmids they were planning to create during this summer. Discussions involved around the correct sequences of the desired genes, promoters, and the backbone plasmids. They then attended more lab training and headed off to UES and spent the remainder of the day designing the plasmids.

## Thursday

### Lab 1:

Colonies successfully grew on plates. We picked colonies and created overnights with LB Broth. 2 overnights were created for each colony. One overnight would include 3 μL of the quorum sensing molecule N-butanoyl-L homoserine lactone (C4-HSL) to induce the production of the GFPa1 and the other overnight would not include C4-HSL, but DMSO, to ensure that C4-HSL was the factor responsible for the production of GFPa1.

### Lab 2:

The team met early on Thursday in order to get passes for lab and to attend a security training seminar. After the seminar the team met with Dr. Chia Hung and finalized the plasmid designs. They decided to use the plasmid pET11a for with bHMO. Other designs were agreed upon and checked and once again the team met at UES in order to discuss design and creation of the plasmids. Finally they began work on the safety forms for the upcoming deadlines.

## Friday

### Lab 1:

The entire team and mentors met today at UES to discuss the project in detail, the meetup we plan on hosting June 22, and medal criteria. Afterwards, the Lab Pats headed back to lab. In lab, the team looked at the tubes from the overnights under the blue light. We had successful results. The blanks were not contaminated, the C4-HSL tubes glowed, and the DMSO tubes did not glow. We also made glycerol stocks out of plate #2 tube #2 of the DMSO (Amy's RhlR stock #7)

### Lab 2:

The RX team showed up to lab in order to create needed lab materials and solutions for the next week's workdays. They created LB Broth and created plates with Amp and Kan resistance. After this they attended an entire team meeting where discussion revolved around the meetup we are planning to hold on June 21, safety forms, and the new emphasis by iGEM on measurement and the project description and inspiration. Additionally, the team discussed in great length the design of the parts that the RX team is planning to make. Discussions at the meeting were very helpful in propelling the team towards completing their massive goals for the summer. IDT orders were sent in for two of the designed constructs.

# 6/10/2019 - 6/14/19

## Monday

### Lab 1:

LabPats composed an LB Broth and Chloramphenicol solution (3mL broth
and 3μL antibiotic). 3 tubes were then made with 3μL C4-HSL and DMSO in
respective tubes. The glycerol stock we made on 6/7/19 was put into
DMSO and C4-HSL only, and the tubes were put into overnights at 37o C

### Lab 2:

To start of the week, the RX team showed up tolab early in the morning in order to complete more of their newcomer training into lab. They then attended a seminar and headed off to complete lab work. They borrowed frozen stock of cells containing pY71-SP and pET11a, plated them out, and then placed them in an incubator to try to grow new colonies of cells. After this the team headed to UES Inc. in order to work on the non-completed constructs since there is still some discussion on what needs to be accomplished for the project. Safety forms and human practices of researching companies were also worked on. In addition to the work at UES, a portion of the team returned to base to create liquid culture of the pY71-SP and discuss safety forms with Chia.

## Tuesday

### Lab 1:

The LabPats miniprepped the RhlR from the overnights. We followed
the miniprep procedure with the goal of extracting the plasmid. The
tubes were labeled RhlR 1 and RhlR 2. LabPats made overnights of
both the DMSO and C4-HSL.Nanodrop Results of C4-HSL Induced RhlR
Plasmid

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 | Tube Number | 260/280 ratio | DNA concentration (ng / μL) |
| 2 | 1 | 1.88 | 196.5 |
| 3 | 2 | 1.88 | 80.3 |
| 4 | EB | 0.42 | 0.1 |

### Lab 2:

Excited to start prepping their plasmids, the RX team showed up to lab and checked out the culture they grew over the previous night. The culture appeared to have grown and they waited until their final lab training to mini-prep 50 mL of culture containing pY71-SFGFP and 50 mL culture containing pET-11a. After the DNA was collected they checked the restriction sites of the plasmids, and performed an overnight digest.

## Wednesday

### Lab 1:

In lab, we made more overnights that included 3 tubes of 3OC12 with
sfGFP (2017 part), 3 tubes of DMSO with sfGFP, 3 tubes of C4-HSL
with RhlR plasmid, 3 tubes of DMSO with RhlR plasmid, and a blank.
The 3OC12 with sfGFP is the LabPats part from 2017 that we are
quantifying this year. For the 3OC12 tubes, the concentration was
3μL of 10μM into 3mL of LB. The C4-HSL concentration was 3μL of
100μM into 3mL of LB. These 13 tubes were put into the incubator at
37o C overnight.The team also met up with 3 members of the USAFA
iGEM team for lunch at City Barbecue.

### Lab 2:

Early in the morning when the RX Lab team showed up to lab, they also ran a gel of the overnight plasmid to verify the presence of their DNA and performed a gel extraction collecting the desired linear pieces of DNA to place into their backbones. Seeing the bands were incorrect, the team set up another overnight digest of pY71-SFGFP and pET-11a.

## Thursday

### Lab 1:

Lab Pats made standard curves and took measurements of the
fluorescence and absorbance of the overnights from Wednesday
(6/12/19). We used both the plate reader and flow cytometer to
measure overall fluorescence and absorbance in the wells and
individual cell absorption/fluorescence respectively. We also did a
1/2 dilution in order to fit the measurements in our standard
curves.

Plate Reader

Settings for absorbance: 600 OD, top read, purple adapter for
bottom, 10 seconds shaken, clear wells

Settings for fluorescence: 480-510λ, bottom read, 5 seconds shaken,
black well w/ clear bottom

Green to white (A1-D12) serial dilutions for standard curve

Dark red (E1-E3) RhlR w/C4HSL

Light red (E4-E6) diluted RhlR w/C4HSL

Dark Orange (F1-F3) RhlR w/DMSO

Light orange (F4-F6) diluted RhlR w/DMSO

Dark blue (G1-G3) pLas w/ 3OC12

Light blue (G4-G6) diluted pLas w/3OC12

Dark purple (H1-H3) pLas w/DMSO

Light purple (H7-H9)

Black (H4-H6 and (H9-H12)

![](https://static.igem.org/mediawiki/2019/b/b1/T--US_AFRL_CarrollHS--Table_6-13-2019.png)

Flow Cytometer

LabPats used spherotech cat. no. URCP 38-2k log no. Ak02

300 μL of culture were in the tubes and then 1 mL of 1X PBS buffer
was added to the tubes

There were 13 tubes total with 100 μL per sample. 1-3 was C4HSL in
RhlR plasmid. 4-6 was DMSO in RhlR. 7-9 was 3OC12 in pLas. 10-12
was DMSO in pLas. 13 was the beads provided in the kit (1-2
drops)We also made a 150mL overnight from glycerol #7 of the RhlR
plasmid in preparation for the maxiprep on 6/14.

### Lab 2:

Heading towards the end of the week, the RX team began their day by running a gel with the digested pY71-SFGFP and pET-11a in order to verify correctly cut bands since the one performed the previous day did not work. Also they performed a gel extraction and purification of the desired DNA. Once they obtained the DNA, they were taught how to use the QuBit machine to check the exact concentration of their DNA. While the gel extraction was being performed, some of the RX lab students mini-prepped 50 mL of Rhl-R7 culture provided by MIke and the RH team.

## Friday

### Lab 1:

The LabPats took RhlR overnights from 6/13 out of the incubator and
did a MaxiPrep in order to prepare the DNA for cell-free reactions.
We used the ZYMO kit and followed the MaxiPrep procedure.

Nanodrop Results of C4HSL Induced RhlR Plasmid (Maxiprep #1)

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | EB | RhlR |
| 2 | Ratio (260/280) | 1.13 | 1.85 |
| 3 | Concentration (ng/μL) | -1.3 | 79.9 |

### Lab 2:

The team ended the last day of the week by starting their day at UES. They were working on safety forms and human practices outreach and ideas. They then headed to lab to converse with Chia about the new part they had to develop after having a conversation with MIke. After the productive meeting, they returned to UES to finish out the day by designing two G-Blocks needed to further the project.

# 6/17/2019 - 6/21/2019

## Monday

### Lab 1:

We made an overnight today for another Maxiprep. It was also taken
from glycerol stick #7 (A.E.), but this time was made with200 mL of
LB broth and 200 μL of chloramphenicol. This overnight was put in
at 37o C overnight.Then LabPats made overnights of 6 different
stocks of 3OC12 in pLas plasmid (the sfGFP 2017 part) in order to
find the one that fluoresced to be used for the characterization of
an existing part. There were pLas B5, pLas from 6/29/17, pLsr D5,
pLsr F2, pLsr E0, and pLsr D2. We also made overnights of their
DMSO and a blank that had 3μL of chloramphenicol per 3 mL of LB.
There were 13 tubes total which were put in the incubator at 37o C
overnight.

### Lab 2:

At the start of a new week the team ran a PCR on C4-HSL plasmid from our mentor Mike. The PCR was ran on a gradient to test to see which temperatures would work the best for the following day to purify the PCR then. The students want to amplify the C4-HSL to insert bFMO once the G-Block comes in from IDT. After the working hard in the lab the students worked on the human practices.

## Tuesday

### Lab 1:

We checked the overnights and they did not glow because pLsr does
not glow (wrong plasmid) and either of the pLas we did use were not
the one that worked in 2017. Therefore we made more overnights (3μL
chlor. per 3mL LB) and 3μL of 3OC12 in pLas plasmid (sfGFP) along
with their corresponding DMSO and a blank (15 tubes total).

1. pLas\_sfGFP miniprep TD#1
2. pLas\_sfGFP miniprep TD#2
3. pLas\_sfGFP miniprep TD#3
4. pLas\_sfGFP #6 JD
5. pLas\_sfGFP #3 HJ
6. pLas\_sfGFP #5 JD
7. pLas\_sfGFP #4 HJ

Another maxiprep of RhlR was done but with some modifications in
order to try and increase the DNA amount. The team used 200mL of
RhlR instead of 150mL, heated the EB to 50o C, used 200 μL of EB
instead of 400L, and let the EB sit for 10 minutes prior to
spinning down.

Nanodrop Results of Maxiprep #2

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | EB | C4HSL |
| 2 | ratio 260/280 | 0.88 | 1.84 |
| 3 | concentration (ng/μL) | 0.5 | 79.7 |

### Lab 2:

The new day begins with running the PCR on a gel and analyzing to see which bands were the strongest to redo the reaction at one temperature. After setting up another PCR reaction of C4-HSL and running that on a gel the RX Lab Students found a similar result of the PCR the day before, but with stronger bands. The boys purified the PCR and waited for bFMO come in later that week.

## Wednesday

### Lab 1:

One of the overnights fluoresced pLas\_sfGFP\_JD #5. The LabPats made
triplicates with this plasmid along with the corresponding DMSO and
a blank. Then we made an ethanol precipitation of the two RhlR
MaxiPreps from 6/14 and 6/18 in order to increase the concentration
of DNA.

MaxiPrep Results after Ethanol Precipitation

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | ratio 260/280 | concentration ng/mL |
| 2 | #1 (6/18) | 1.83 | 87.1 |
| 3 | #2 (6/14) | 1.87 | 243.1 |
| 4 | EB | -0.4 | 0.62 |

For measurements, wells E-G 5-11 were the dilution for sfGFP (green
to white) and wells B-D 5-11 were the dilution for fluorescence
(blue to white). The C4-HSL dilution was 20 fold (.5μL of C4HSL and
9.5 μL of DMSO).

![](https://static.igem.org/mediawiki/2019/5/54/T--US_AFRL_CarrollHS--_New_Table_6-19-2019.png)

Wells B2-B4

negative control (red)

10μL premix

9μL extract

6μL water

Wells C2-C4

positive control (purple)

10μL premix

9μL extract

3μL DNA (RhlR)

3μL water

Wells D2-D4

DMSO w/o polymerase (light green)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL DMSO

1μL water

Wells E2-E4

C4HSL w/o polymerase (light blue)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL C4HSL

1μL water

Wells F2-F4

DMSO w/polymerase (E. coli RNA Polymerase Holoenzyme S M0551S from
New England Biolabs) (yellow)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL DMSO

1μL polymerase

Wells G2-G4

C4HSL w/polymerase (E. coli RNA Polymerase Holoenzyme S M0551S from
New England Biolabs) (orange)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL C4HSL

1μL polymerase

The Promega Kit (S30 T7 high-Yield Protein) Expression System was
used for these cell-free reactions. The master mix protocol was
followed (see above) and put in a 96 well plate. The plate was
covered with a non breathable membrane. MaxiPrep #1 from 6/18 was
used. The plate (REF L1110) was in the plate reader overnight.

Settings for fluorescence in plate reader: 480-510λ, bottom read
every 10 minutes for 12 hours at 30oC, 5 seconds shaken, black well
w/ clear bottom

### Lab 2:

The students IDT order came in for bFMO and chrB then there was something for the boys to do. Then the students assembled PY71-bFMO and pET-11a-chrB using the Gibson assembly method. The students then transformed on to plates hoping for colonies the following day. They also did a PCR of bFMO on a gradient to be put in with C4-HSL PCR done the previous day.

## Thursday

### Lab 1:

Today the LabPats measured the 2017 iGEM part. This began with a
dilution of the cultures (50μL of LB and 50μL of culture). A1-A3
were samples of 30C12 JD #5, A4-A6 were samples of DMSO, and A7 was
the blank in a 96 well plate.Plate Reader

Settings for absorbance: 600 OD, top read, purple adapter for
bottom, 10 seconds shaken, clear wells, 22.7oC Settings for
fluorescence: 480-510λ, bottom read, 5 seconds shaken, black well
w/ clear bottom, 22.7oC

### Lab 2:

The RX Lab Student ran a gel on the C4-HSL PCR and bFMO to look for proper band location. The students decided to a gradient on the bFMO to try to get strong bands and also redo the C4-HSL at a different temperature hoping for better bands. The pET-11a-chrB transformation was successful with nine total colonies, but the PY71-bFMO had no colonies so the students redid the transformation hoping for better results the next day. Chia also believed to that the students should try to use blunt end to assemble bFMO into TOPO so bFMO could be cute out and assembled into PY71.

## Friday

### Lab 1:

The LabPats prepared for their meetup on Saturday.

### Lab 2:

Going into the last day of the week the students digested pET-11a-chrB with EcoR1 trying to verifiy that chrB has successfully been cloned in to pET-11a. Ran PCR of bFMO trying to get a better band to put into C4-HSL. Digested some more of the PY71 -GFPa1 and pruifeid the PY71 because the students were running low on some of that DNA. Finally the students went back to work on the presentation for the meet up that weekend.

# 06/24/2019 - 06/28/2019

## Monday

### Lab 1:

Two bottles of LB were made. It was in the autoclave for 15 minutes
with the flashing sign/liquid cycle. We made sure that the bottles
were not too tightly sealed and had autoclave tape on them. The
bottles were labeled LB broth iGEM 6/24/19.

### Lab 2:

Beginning the new week, the team began their day in lab where they checked on the growth of the plates containing TOPO-bFMO and found cells. Afterwards the cells were inoculated in LB Broth with a Kan resistance picking 16 colonies, half of which appeared as indigo on the plate and the other half of which appeared as white. Also they ran two gels, one to check the PCR that was ran over the weekend and the other for the digested pET11a-ChrB in order to cut and purify. Once lab work for the day was completed, the team headed to UES in order to work on visuals for the presentation, safety forms, and human practices.

## Tuesday

### Lab 1:

An overnight of RhlR for a new MaxiPrep was made. A sterile bottle
was used since we did not have sterile flasks. The solution
included 200mL of LB Broth, 200μL of chloramphenicol, and RhlR
stock #7 A.E. It was placed in the incubator at 37oC overnight (we
made sure the cap wasn't too tight)

### Lab 2:

The new day began with the RX Lab Students running a gel on the mini prepped 16 samples of TOPO-bFMO and they chose colonies 1,2,5,7,8 for further experimentation. Also, they ran a PCR of the bFMO fragment. After the PCR was completed, they ran a gel that was purified. Finally they started an overnight digest of pET11a-sfGFP and left it overnight for the following day.

## Wednesday

### Lab 1:

The MaxiPrep of the overnight from 6/25 was completed.

Nanodrop results from MaxiPrep of RhlR

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | ratio 260/280 | concentration ng/ml |
| 2 | RhlR | 1.89 | 3381.5 |
| 3 | EB | 1.48 | -0.6 |

The cell-free reactions were then set up using the DNA from the
MaxiPrep. The same protocol was followed as on 6/19 but with 0.6μL of
DNA for each reaction (DNA Calculation 0.6μL \* 3382 = 2000ng so we want
2μg of DNA). Therefore, the adjusted reactions were as follows:

B2-B4, negative control

C2-C4, positive control

D2-D4, DMSO w/o polymerase, 0.6μL DNA, 4.9 μL H2O

E2-E4, C4HSL w/o polymerase, 0.6μL DNA, 4.9 μL H2O

F2-F4, DMSO, 1μL polymerase, 0.6μL DNA, 3.9μL H2O

G2-G4, C4HSL, 1μL polymerase, 0.6μL DNA, 3.9μL H2O

Plate reader settings: endpoint reading for fluorescence, read from the
bottom, 480-510λ, sensitivity 19, autoread 600, temperature 30o C.

### Lab 2:

Wednesday proved to be one of the busiest days the team has experienced in a while beginning with running a digest on the TOPO-bFMO plasmid. Additionally, they ran a gel of pET11a and cut and purified the vector of the plasmid desired for assembly. They then performed an assembly with chrB and transformed into NEB-5 Alpha. While this was going on, the team ran a PCR on the bFMO fragment. After seeing the purification of the bFMO from the previous day was unsuccessful, they ran a gel, cut and purified the new PCR of the bFMO fragment and obtained a very high concentration that will be used for a future assembly with C4-HSL.

## Thursday

### Lab 1:

The LabPats did a data analysis of the measurements from the plate
reader. The data showed that the RhlR circuit #2 did not work. So we
decided to test with the S30 E.coli (Extract for circular plasmid)
native kit. This is a Promega Kit (cat. # L1020). This cell free
reaction has more steps to the protocol than previous 25μL reactions,
including complete amino acid for all the reactions (which is a mixture
of amino acid without methionine and amino acid without leucine) and
synthesis and assays of luciferase for the positive control.

Reaction A -

No DNA: wells B2-B4

DMSO: wells D2-D4

10μL Premix- 7.5μL Extract- 2.5μL complete amino acid (RhlR)5μL- nuclease
free water

Reaction B -

pY71: wells C2-C4

10μL Premix- 7.5μL Extract- 3μL DNA (pY71-sfGFP)- 1μL T7 Polymerase- 2.5μL
amino acid

Reaction C -

10μL Premix- 7.5μL- 0.6μL DNA- 0.5μL DMSO- 2.5μL amino acid- nuclease free
water

Reaction D -

C4-HSL: wells E2-E4

originally 10μL Premix

prior to

7.5μL Extract- 0.6μL DNA (RhlR)- 0.5μL C4-HSL- 2.5μL amino acid- 3.9μL
nuclease free water- 1μL nuclease free water- 3.9μL

Blank -

wells F2-F4

There is nothing in these wells because they weresupposed to be luciferase,
but we did not incubate (step 3 in protocol) starting reactions B-D.
![page9image41230592](blob:https://wordhtml.com/d773d08e-e7e9-4669-890a-ba221a63316e)

### Lab 2:

Arriving to lab on Thursday, the RX students found a few colonies growing on their transformation of pET11a-chrB, picked 2 viable colonies and set up the culture to grow overnight. Additionally they ran a gel on the on the bFMO-TOPO plasmid, cut and purified the bFMO.

## Friday

### Lab 1:

Today's focus was on redoing the luciferase control because it was
not measured on Thursday as planned. There were two reactions: the
luciferase and no DNA. The luciferase required incubation for an
hour at 37o C, an ice bath for 5 minutes, and a dilution series
before being pipetted into a 96 well plate. It was originally done
in wells G1-8 and H1-12 (G1-G4 had the stock solution, G5-G8 had
the 1st dilution, H1-H4 had the 2nd dilution, and H9-H12 had no
DNA). Then the experiment was redone. now with B2-B5 having the
stock solution, B6-B9 had the 2nd dilution, C2-C5 had the 3rd
dilution, C6-C9 had the 4th dilution, and D2-D5 had no DNA.

Luciferase Reaction

2μL pBESTluc DNA2.5μL complete amino acid mix- 10μL Premix- 7.5μL
Extract- 3μL Nuclease free water

No DNA Reaction 10μL Premix- 7.5μL Extract- 2.5μL Amino acid- 5μL
Nuclease free water

### Lab 2:

The end of the week snuck up on the RX Lab students as they prepared to accomplish as much as possible for over the weekend. Colonies 1 and 2 of pET11a-chrB were mini prepped from the culture created the previous day. They ran a small digest and gel and found that the gel showed the correct size bands for pET11a-chrB and 20 mL of culture was grown up on Sunday to prepare for Monday. Additionally, C4-HSL-bFMO was assembled and transformed and left to grow on the bench over the weekend. Finally a ligation was created for over the weekend of pY71-bFMO. Outside of lab, the students met with Scott Lanter who was an airplane painter for 35 years for the Air Force. He shared his knowledge and experience of the chromium paint and safety processes involved with it all.

# 7/1/2019 - 7/5/2019

## Monday

### Lab 1:

The LabPats lyophilized three reactions: one with no DNA, one with
pY71-sfGFP added before being rehydrated and one with pY71-sfGFP added
after being rehydrated (it was resuspended with DNA and water). The
purpose of this was to see if functionality was improved with DNA being
added before or after. For each (no DNA, pY71 before, and pY71 after),
we did a 25μL reaction and four 5μL reactions. These reactions froze in
the -80o freezer for 10 minutes and then in liquid nitrogen for 1
minute. After this, the caps were removed and put in the lyophilizer
overnight in order to remove the liquid.

No DNA reaction 10μL Premix- 9μL Extract- 6μL Water

pY71-sfGFP DNA after- 10μL Premix- 9μL Extract- 6μL Water- (Resuspended
with DNA and water)

pY71-sfGFP with DNA before 10μL Premix- 9μL Extract- 3μL pY71-sfGFP-
3μL Water

### Lab 2:

Hopes were high for this week in lab where the team was planning on successfully assembling, transforming, and verifying three different parts in order to be able to start the process of the RH Lab Students to make them work in a cell free system. Throughout the day the students worked on mini-prepping 20 mL of NEB-5 Alpha pETlla-chrB Chia made on Sunday and transforming the prepped plasmid into BL21 (DE3). Additionally, they performed a digest and ran a gel to verify the correct size of pET11a-chrB and made freezer stocks of the cultures containing NEB-5 Alpha pETlla-chrB. Also, they transformed the ligation of pY71-bFMO started on Friday into NEB-5 alpha. Finally they transformed C4-HSL-bFMO into NEB-5 Alpha since the transformation from Friday did not work since it was found that the wrong antibiotic LB Agar Plates were used. During the middle of the day, they had a whole team meeting with Diane Buhrmaster who explained to them the military and government regulations and safety protocols involving the use of hexavalent chromium.

## Tuesday

### Lab 1:

The reactions from Monday (7/1) were rehydrated and run through the
plate reader, showing that the lyophilization was successful.
Reactions 1 and 2 were rehydrated with the same amount of water as
the reaction (so the 25μL rxn was rehydrated with 25μL of water and
5μL water with 5μL reactions). Reaction 3 had both water and
pY71-sfGFP DNA that was rehydrated in 3μL DNA and 22μL water. We
took pictures of the plate for visual proof of fluorescence.Since
our cell free reactions had not been successful, LabPats began
troubleshooting. We redid our cell free reactions in both T7 and
S30 kits, but with the native control pAME 215. pAME 215 is a
strong, modified native control which we used as a replacement for
pY71-sfGFP since it didn’t work previously in the S30. We did these
in 5 μL reactions with no DNA, pAME 215, or pY71-sfGFP in both
kits.

T7 No DNA (B2-5 and D2-5)

10μL Premix

9μL Extract

6μL Water

1μL Water

S30 No DNA (C2-5)

10μL Premix

7.5μL Extract

2.5μL Complete amino acid mixture

5μL Water

T7 pAME 215 (B6-9)

10μL Premix

9μL Extract

5μL Native control

1μL Water

S30 pAME 215 (C6-9)

10μL Premix

7.5μL Extract

5μL pAME 215

2.5μL Complete amino acid

T7 pY71-sfGFP (E2-5)

10μL Premix

9μL Extract

5μL pY71-sfGFP

Then we did an experiment to test if DMSO was having an effect on
the cell free reactions. We used the T7 kit for this and had
reactions of no DNA, pY71-sfGFP + DMSO, and just pY71-sfGFP.

No DNA

10μL Premix

9μL Extract

6μL Water

pY71 + DMSO

10μL Premix

9μl Extract

3μL pY71-sfGFP

0.5μL DMSO

2.5μL Water

pY71

10μL Premix

9μL Extract

3μL pY71-sfGFP

3μL Water
![page10image41632320](blob:https://wordhtml.com/1eda3882-4053-4106-a4e9-5e4eb8b18495)

### Lab 2:

Excitement caught the team as they entered lab and discovered that each and every one of their three transformations from the previous day had produced colonies. After choosing colonies to inoculate, they created 12 cultures that will grow overnight: 2 colonies of pET11a-chrB in BL21 (DE3), 5 colonies of pY71-bFMO in NEB-5 Alpha, and 5 colonies of C4- HSL-bFMO. They also met with Chia and finalized two G-Blocks they plan to order from IDT (chrP-bFMO and chrP- GFPa1). The last thing they accomplished before leaving lab was prepping pTOPO-bFMO and pET11a-chrB and sending it out for sequencing. After all the lab work was completed the RX Lab students headed to UES where they worked on the presentation they were going to use at the MIchigan State meet-up for the following weekend, contacting companies for human practices, and catching up on lab notes and all other minor project details.

## Wednesday

### Lab 1:

Troubleshooting continued today with DNA titrations. We had been
doing our cell free systems with a final concentration of 80ng/μL,
and we were advised that we may be overloading the system with this
amount of DNA. With this is mind, we did our titrations with 20,
40, 60, and 80ng/μL of DNA along with corresponding DMSO. Then we
put the reactions in the plate reader overnight.

Reaction A (No DNA) B2-B5

10μL Premix

9μL Extract

6μL Water

Reaction B (+ control pAME215) B7-B10

10μL Premix

9μL Extract

5μL DNA

1μL Water

Reaction C (80ng/ μL with DMSO) C7-C10

10μL Premix

9μL Extract

0.6μL DNA (RhlR)

0.5μL DMSO

Reaction D (80ng/μL)

C2-C5

10μL Premix

9μL Extract

0.6μL DNA (RhlR)

0.5μL C4HSL (5mM)

4.9μL Water

9μL Extract

5μL DNA

1μL Water

Reaction E (60ng/μL with DMSO)

D7-D10

10μl Premix

9μL Extract

4.4μL DNA (RhlR)

0.5μL DMSO

1.1μL Water

9μL Extract

0.6μL DNA (RhlR)

0.5μL DMSO

Reaction F (60ng/μL)

D2-D5

10μL Premix

9μL Extract

4.4μL DNA (RhlR)

0.5μL C4HSL(mM)

1.1μL Water

Reaction I (20ng/μL with DMSO)

F7-F10

10μL Premix

9μL Extract

1.5μL DNA (RhlR)

0.5μL DMSO

4μL Water

Reaction G (40ng/μL with DMSO)

E7-E10

10μL Premix

9μL Extract

5μL DNA (RhlR)

0.5μL DMSO

0.5μL Water

Reaction J (20ng/μL)

F2-F5

10μL Premix

9μL Extract

1.5μL DNA (RhlR)

0.5μL C4HSL (5mM)

4μL Water

Reaction H (40ng/μL)

E2-E5

10μl Premix

9μL Extract

5μL DNA (RhlR)

0.5μL C4HSL (5mM)

0.5μL Water

### Lab 2:

Starting off the day in lab, the RX Lab Students began with a mini-prep of pY71-bFMO and C4-HSL-bFMO. Once the mini- prep was completed, an overnight digest was set up for both of the plasmids. Also, a protein gel was set up for pET11a- chrB in order to determine if chrB was in fact being produced. After the lab work was completed, the RX Lab Students headed to UES where they worked on human practices and write up for meetings.

## Thursday

### Lab 1:

LabPats were off for 4th of July.

### Lab 2:

Day taken off for the Holiday!

## Friday

### Lab 1:

LabPats were off for the 5th of July.

### Lab 2:

Ending off the week the RX Lab Students came into lab with only one thing they hoped to accomplish. They took the spun down induced and uninduced colonies of pET11a-chrB from Wednesday and began the process to run them onto a protein gel. After all of the protocols were completed, they looked at the gel and found that the induction had not worked as intended so a plan was made for another induction to be set up on the following Monday.

# 7/8/2019 - 7/12/2019

## Monday

### Lab 1:

We received new plasmids from RX. They are pY71-bFMO, C4HSL-bFMO, and
pET11a-ChrB. We followed the miniprep protocol to miniprep the pY71-bFMO
which were labeled pY-bFMO 1 and pY-bFMO 2. pY71-bFMO has resistance to the
antibiotic kanamycin.

MiniPrep of pY-71bFMO

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | concentration | ratio |
| 2 | miniprep #1 | 53.3 | 1.83 |
| 3 | miniprep #2 | 63.8 | 1.85 |
| 4 | EB | 0.0 | 1.04 |

Then we used miniprep #2 for the transformation. The plates were
labeled pY71-bFMO BL21-DE3 1X and pY71-bFMO BL21-DE3 9X. BL21-DE3 are
competent E. coli cells. The plates were incubated overnight.We also
followed the maxiprep protocol with both the old and new kits in order
to maxiprep all three of the plasmids, and we made glycerol stocks of
them. The maxiprep for pY71-bFMO was spilled during the procedure so we
could not get data from it.

Maxiprep for new plasmids

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | Concentration ng/μL | ratio 260/280 |
| 2 | pY-bFMO #1 | no data | no data |
| 3 | pY-bFMO #2 | no data | no data |
| 4 | C4HSL bFMO #1 | 52.0 | 2.00 |
| 5 | C4HSL bFMO #2 | 37.1 | 1.81 |
| 6 | pET11a-ChrB #1 | 50.2 | 1.95 |
| 7 | pET11a-ChrB #2 | 60.6 | 1.93 |
| 8 | EB | -0.3 | 0.69 |

We made 200mL overnights of each plasmid with 200μL of the
corresponding resistance. For pY71-bFMO that is Kanamycin, for
pET11a-ChrB that is Ampicillin, and chloramphenicol for C4HSL-bFMO. We
induced C4HSL-bFMO sterile with 5μL of C4HSL and also made 3 overnights
of C4HSL-bFMO using the glycerol stock that was made earlier and 3μl of
C4HSL, 3 other overnights with 3μl of DMSO and 1 blank consisting of
3mL of LB Broth.

### Lab 2:

Hopes were high as the new week started with sequencing results arriving over the weekend. The RX Lab students began their day by meeting with Chia and discussing the plans for the week. Sequences of pET11a-chrB came back and verified that they had successfully transformed the chrB into the plasmid. They delivered the overnight 500 mL of cultures of the three completed constructs to the RH Lab Students so that they could start to work with them and attempt to make them function in a cell free system. Also the team transformed pY71-bFMO into BL21 (DE3) while starting a new overnight induction of pET11a-chrB for protein expression. Finally they started an overnight culture of PSB3K3 given by Mike. After all of the lab work was completed, the students headed to UES where they worked on the Michigan State Meet Up presentation and contacting companies for Human Practices.

## Tuesday

### Lab 1:

LabPats followed the maxiprep protocol for each of the 200μL overnights
from yesterday.

Maxiprep for overnights of bFMO and ChrB

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | concentration ng/μL | ratio 260/280 |
| 2 | pY71-bFMO | 184.7 | 1.9 |
| 3 | pY71-C4HSL | 709,1 | 1.87 |
| 4 | pET11a-ChrB | 910.4 | 1.88 |
| 5 | EB | -0.8 | 0.76 |

Then we redid the transformations since the ones from yesterday
were not successful. The overnights from last night did not express
bFMO so we made new overnights but this time with tryptophan added.
We did not use straight tryptophan but a recipe of it dissolved in
water. The recipe was 200μL of milli-q water and 1g of tryptophan
which is 5mg/μL. For pY71-bFMO, there was one blank with 3mL of LB
broth and 3μL Kan, 3 tubes of pY71-bFMO with 3mL of LB broth and
3μL Kan, and 3 tubes of pY71-bFMO with tryptophan added. These last
tubes had 2.4mL of LB, 600μL of tryptophan, and 3μL Kan. The same
was for the C4HSL-bFMO except the antibiotic was chloramphenicol.

### Lab 2:

The RX Lab students began their day by making a new protein gel of pET11a-chrB, but this time using 2x BME dye instead of 4x LDS sample suffer. While the new protein gel was being prepared, the overnight culture of pSB3K3 was mini- prepped and colonies of pY71-bFMO in BL21 DE3 were picked and grew up.

## Wednesday

### Lab 1:

We checked the overnights and saw that some of the pY71-bFMO with
tryptophan had expression of bFMO. However, none of the C4HSL-bFMO
tubes expressed bFMO. We looked at the transformation and there
were tiny colonies and a film on the plates so we redid the
transformation using the same protocol from 7/9/19 except using 50
Kam plates instead of 25 Kam. We streaked three plates for each
transformation (at 1X and 9X concentration).Then the LabPats did
cell free reactions with pY71-bFMO since cell free reactions with
C4HSL were not working. This DNA was tested alone and then with
tryptophan, with DMSO, and with both in quadruplicates. The plates
were put in the plate reader overnight with the absorbance set at
620nm and an endpoint reading for the positive control (pY71-sfGFP)
was taken the next morning.

![page13image41660032](blob:https://wordhtml.com/134b06d4-c17e-4895-b0fa-da5cc4e8f643)

RXN A (No DNA)

Wells B2-B5

10μL Premix

9μL Extract

6μL Water

RXN B (pY71-sfGFP)

Wells B6-B9

10μL Premix

9μL Extract

3μL Water

3μL pY71-sfGFP

RXN C (pY71-bFMO)

Wells C2-C5

10μL Premix

9μL Extract

2.8μL Water

3.2μL pY71-bFMO

RXN D (pY71-bFMO +Trp)

Wells C6-C9

10μL Premix

9μL Extract

3.2μL pY71-bFMO

2.3μL Trp

0.5μL Water

RXN E (pY71-bFMO +DMSO)

Wells D2-D5

10μL Premix

9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO

2.3μL Water

RXN F (pY71-bFMO +both)

Wells D6-D9

10μL Premix

9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO

2.3μL Trp

### Lab 2:

At the start of the next day, the RX Lab students focused on spending their day to continue distaining of the protein gel while preparing another chrB protein gel by inducing a culture of chrB and running it on a new protein gel. In addition to running the protein gels, the IDT orders for the G-Blocks of chrP-GFPa1 and chrP-bFMO were delivered to the lab. After receiving the G-Blocks, they were assembled into pY71 and the restulting assembly was transformed into E. coli NEB 5- Alpha.

## Thursday

### Lab 1:

The transformations from 7/10 were successful so we made overnights
from them. The results of the cell free system showed visual blue
at the bottom of some of the wells and the data analysis showed
slightly higher absorbance readings for pY71- bFMO by itself, with
DMSO and with Tryptophan compared to No DNA and pY71-bFMO with both
DMSO and Tryptophan. We have been using tryptophan in the past
experiments because tryptophan is converted into indole by E.coli
which reacts with bFMO to express an indigo color. With us putting
tryptophan into the reactions, it was expected that more indigo
would be expressed. With positive results from the last overnights
with Trp, we decided to make overnights with tryptophan, IPTG
(which is supposed to induce T7 polymerase allowing for greater
production from our plasmid), both Trp and IPTG, and neither. These
overnights were quadruplicates made from 4 different colonies,
resulting in 17 overnights including the blank.

Cells as seen in the table means 20μL LB and one of the colonies
(1, 2, 3, or 4) that was then aliquoted into four 5μL tubes in
order to be put into A, B, C, and D overnights (see left)

A = with IPTG

B = neither

C = with IPTG and Trp

D = with Trp

Recipe for overnights with and without Trp and IPTG

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | A | B | C | D | E | F |
| 1 | Label | Cells | Trp (final concentration1mg/mL) | 1M IPTG (final concentration0.5mM) | LB | 50 KAN |
| 2 | A | 5μL | - | 1.5μL | 3mL | 3μL |
| 3 | B | 5μL | - | - | 3mL | 3μL |
| 4 | C | 5μL | 600μL | 1.5μL | 3mL | 3μL |
| 5 | D | 5μL | 600μL | - | 3mL | 3μL |
| 6 | Blank |  |  |  |  |  |

### Lab 2:

Once again beginning the new day, the new protein gel of chrB was continued to be distained. While this protocol was being followed, experiments of BL21 DE3 pY71-bFMO were being set up ranging the amounts of tryptophan, an amino acid found in the literature to be what is converted by the bFMO gene into the indigo color, to find which amount of tryptophan is optimal to turn the pY71-bFMO culture the deepest indigo color possible. Also, a final chrB protein gel was begun with a new protocol to separate the cell pellet from the cell lysate since it was hypothesized that the chrB protein was clinging to the cell pellet.

## Friday

### Lab 1:

LabPats checked the overnights. Group D tubes (with tryptophan)
expressed a lot of indigo and were very dark. This confirmed that
bFMO works in cells. We also saw that IPTG seemed to kill the
reaction since tubes with IPTG in them weren't as dark (both with
and without Tryptophan). Colony 4 did not grow in reactions A and
C. We made glycerol stocks labeled B1- B4 since B was the label for
overnights without IPTG and Trp.In order to measure the amount of
indigo expressed by bFMO, we must extract the indigo using DMSO and
spin the pellet down since indigo and cells have the same
absorbance, and therefore simply putting the overnights in the
plate reader would not give us the amounts of indigo. While we knew
how to do the extraction protocol, we were not sure of the correct
amount of overnight or DMSO needed for our supernatant to not be
too diluted but also remove as much of the indigo from the pellet
as possible. This led to a lot of trial and error.

![page14image41492352](blob:https://wordhtml.com/9e13a77a-aa7b-4475-99cf-13ab0f69b60e)

We began by placing 1000μL of overnight into a 1.5μL tube except
for those with colony 4 which did not have much growth if at all.
These were spun down in the centrifuge at 13,400 rpm xg for 5
minutes. We took out the supernatant, careful not to remove the
pellet with it. Then we resuspended with 500μL of DMSO. When this
did not yield the results we wanted, we experimented with
extraction volumes.Samples A2 and A3. 10μL, spin, add 10μL DMSO,
spin, add 20μL, spin, add 60μL, spin, remove into separate tube,
add 200μL, spin, remove and combine with supernatant in separate
tube, add 200μL. For A2, add another 20μL, spin, remove to separate
tube, add 20μL, spin, remove to separate tube, add 20μL, and remove
to separate tube.Samples D2 and D3: 500μL, spin, remove to separate
tube, add 500μL, remove to separate tube, add 1mL, spin, remove to
separate tube, add 1mL, spin, remove to separate tube, add 1mL,
spin, remove to separate tube, and 20μL and spin down.Samples D1
and A1: 500μL, spin, For A1: remove to separate tube, add 500μL,
For D1: remove to separate tube, add 1mL, spin down, remove to
separate tube, add 1mL, spin, remove to separate tube, add 500μL,
spin down.Samples B1-B3 and C1-C3: 500μL, spin, separate into a new
tube, add 500μL, spin, separate, add 500μL. Then, we ran a few of
these on the plate reader at absorbance at 620nm.

bFMO Extraction

Table1

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 | Plate and Volume | DMSO Abs. (a.u) | pY71-bFMO Abs. (a.u) |
| 2 | V-bottomed,5ul | 1.009 | 0.992 |
| 3 | V-bottomed, 20 μL | 1.293 | 1.413 |
| 4 | Flat, 100 μL | 0.039 | 1.189 |
| 5 | Flat, 25 μL | 0.036 | 0.114 |
| 6 | Flat, 50 μL | 0.033 | 0.451 |

Table2

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | A | B | C | D |
| 1 | Flat, 100 μL | Well #1 Abs. (a.u) | Well #2 Abs. (a.u) | Well #3Abs. (a.u) |
| 2 | pY71-bFMO + IPTG | 0.432 | 0.661 | 0.564 |
| 3 | pY71-bFMO | 0.691 | 0.597 | 0.686 |
| 4 | pY71-bFMO + Trp + IPTG | 0.912 | 0.296 | 0.458 |
| 5 | pY71-bFMO + Trp | 1.153 | 1.116 | 1.017 |

Table3

|  |  |  |
| --- | --- | --- |
|  | A | B |
| 1 | Flat, 100 μL | Well #1 |
| 2 | Empty Well | 0.135 |
| 3 | Water | 1.016 |

### Lab 2:

Coming to the end of the week, the RX Lab Students spent their day by checking the OD values of the pY71-bFMO and running full spectrum scans in order to determine the wavelength value of the indigo color produced by the bFMO gene. The wavelength value appears to be around 670 nm. Also cultures were started for the successfully transformed assembly of both chrP-bFMO and chrP-GFPa1.

# 7/15/19 - 7/19/2019

## Monday

### Lab 1:

Monday morning consisted of preparing for our lab meeting presentation.
In the afternoon, we redid overnights of pY71-bFMO in triplicates with
3mL of LB and 3μL Kanamycin and the glycerol stock B1 from 7/12/19.

RXN A: pY71-bFMO

RXN B: pY71-bFMO + 600μL TrpRXN C: pY71-bFMO + 1.5μL IPTG

RXN D: pY71-bFMO + 600μL Trp + 1.5μL IPTG RXN E: 100μL BL21 DE3
competent cells Blank: 3mL of LB

### Lab 2:

Starting the week, the RX Lab Students made a plan of what all they wished to accomplish for the upcoming week. Once the plan was made, they checked on their protein gel of pET11a-chrB that was left to destain over the weekend. The gel showed that the new process appears to have worked and the correct size band for the chrB protein was produced, adding additional verification the part. They also started culture to begin the induction process of pY71-bFMO, but the culture did not grow to the correct of OD of 0.600 so another overnight culture was set up in order to perform the experiment the following day. Additionally, they mini-prepped the cultures of pY71-chrP-bFMO and pY71-chrP-GFPa1 that grew over the weekend, set up a small two hour digest, and ran it out on a gel. The gel showed three bands when only two were expected, so the team went home for the night and researched what could have happened that night.

## Tuesday

### Lab 1:

Today LabPats redid the bFMO extraction, this time taking 50μL of each
overnight, spinning it down at 13,400g for 5 minutes, discarding the
supernatant, adding 500μL of DMSO, spinning it down again, and removing
the supernatant to a different tube to be measured. Then, to test which
amount gave the most accurate readings, we aliquoted 5μL, 10μL and 25μL
into a 384 well plate and then run in the plate reader. The bL21-DE3
cells were placed directly from the overnight with no extraction
protocol followed. This gave us inaccurate results so we redid the
reading with BL21-DE3 that had been through extraction.

RXN A: Blue

RXN B: Red

RXN C: Yellow

RXN D: Green

RXN E: Black

RXN F: Purple

C2, E2 and G2 were not measured with RXN D because the extracted
supernatant was accidentally thrown into biohaz Rows B and C were 5μL
reactions, Rows D and E were 10μL reactions, and Rows F and G were 25μL
reactions

Absorption Data from bFMO Extraction #2

![](https://static.igem.org/mediawiki/2019/3/3c/T--US_AFRL_CarrollHS--Table_6-19-2019.png)

### Lab 2:

The RX Lab Students went into lab early in the morning in order to start the diluted culture of colonies 1 and 3 of pY71- bFMO for induction/tryptophan testing. While waiting for the culture to grow to an OD of 0.600 in order to start the induction process, they again started another digest of pY71-chrP-bFMO and pY71-chrP-GFPa1, this time leaving it to digest for 5 hours instead of 2 hours since it was hypothesized that the gel from the previous day was incorrect due to an incomplete digest period. Once the culture of pY71-bFMO was induced, the OD was taken at 670.000 nm ( previously determined possible value of indigo produced by bFMO) at hours 1, 2, and 4. Finally once the digest was completed, another gel was ran and bands showed the correct size of both chrP-bFMO and chrP-GFPa1.

## Wednesday

### Lab 1:

We autoclaved empty flasks and bottles using the dry cycle on the
autoclave. Then we did an overnight of the PLas\_sfGFP circuit JD #5. It
included 200mL of LB and 200μL of chloramphenicol.We also did a rehydration
and transformation of TetR, part: BBa\_K876004 (pTET-RBS-GFP-T-pLac-TeTq)
which came from the iGEM Distribution Kit in order to check off the
measurement criteria from the bronze medal. For this transformation we used
2018 plate #2 Well 15N. We punched a hole through the desired well using a
pipette tip, pipetted 10μL of dH2O up and down, and resupspended the DNA
which turned red. Then we transformed the DNA and placed the 9μL that were
left into a tube labeled pTET. LabPats also redid the pY71-bFMO Cell Free
System reactions. They were redone in triplicates and ran overnight on the
plate reader at an absorbance of 620nm. The DNA was at a concentration of
180ng/μL

![page17image41803456](blob:https://wordhtml.com/6e275db3-757d-4a40-94d6-d5984a4fa67e)

RXN A (No DNA)

Wells B2-B4

10μL Premix

9μL Extract

6μL Water

RXN D (pY71-bFMO +Trp)

Wells C2-C4

10μL Premix

9μL Extract3.2μL pY71-bFMO 2.3μL Trp

0.5μL Water

RXN B (pY71-sfGFP)

Wells B5-B7

10μL Premix

9μL Extract

3μL pY71-sfGFP

3μL Water

RXN C (pY71-bFMO)

Wells B8-B10

10μL Premix

9μL Extract

3.2μL pY71-bFMO

2.8μL Water

RXN F (pY71-bFMO +both)

Wells D6-D9

10μL Premix 9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO

2.3μL Trp

RXN E (pY71-bFMO +DMSO)

Wells C5-C7

10μL Premix

9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO2.3μL Water

We also lyophilized which meant making the same 25μL reactions with DNA
as above and putting them in 0.6mL (600μL) tubes. Next, we froze the
reactions in liquid nitrogen for 1 minute. Then we removed the caps and
put the tubes in the lyophilizer overnight to remove the liquid.

The LabPats also made overnights for the 3OC12 Dose Response Curve.
Each overnight had 3mL of LB, 3μL of 3OC12 stock (diluted with
DMSO), and pLas\_sfGFP JD #5. The 3OC12 stocks began with 20μL and
was diluted into 10μL of DMSO down the line of tubes so that each
had a total of 10μL but half as concentrated with 3OC12 as the
previous tube. Then the 10μL were aliquoted into the triplicates so
each had 3μL of the 3OC12. They were then put in the incubator at
37oC.

### Lab 2:

Heading into the middle of the week, the RX Lab Students began their day by transforming the verified plasmids of pY71- chrP-bFMO and pY71-chrP-GFPa1 into BL21 (DE3). Also they collected the final OD readings from the overnight induced culture of pY71-bFMO from the previous day. In addition to the OD readings, they performed a full spectrum OD scan in order to find the wavelength at which the amount that the indigo color produced by bFMO. Finally in lab the students prepared culture of BL21 (DE3) in order to make their own competent cells the following day. Once all of the lab work was completed, the RX Lab Students headed to UES where they worked on Human Practices, on presentations, and catching up on miscellaneous papers and projects.

## Thursday

### Lab 1:

Rehydrating in pr

Maxiprep

Transformation used 2018 kit

Rehydrated Cell free systems because we forgot to put on membrane,
put in incubator for overnight

### Lab 2:

After the RX Lab team entered lab, they picked colonies of the chrP-bFMO and chrP-GFPa1 that were successfully transformed into E. coli BL21 DE3. Also the induction process of pY71-bFMO with full spectrum scans for the presence of indigo produced by bFMO. The scans were performed at hours 1, 2, and 4 after induction with 0.1 uM of IPTG. Later on in the day, the RX Lab Students had a phone call meeting with Jim Martin, a lab manager at the Greene County Waste Water Treatment Plant, in order to discuss chromium in the team's own local area and how they deal with chromium. The use for a biological chromium sensor was also discussed. Finally the whole team went to UES where they had a progress meeting and decided on many things concerning the project as a whole. During the meeting it was discovered that the chrB G-Block that was assembled into pET11a was reversed in its orientation so new chrB primers and a new chrB G- Block was ordered.

## Friday

### Lab 1:

Got endpoint reading for plate

Made LB broth

5μL standard curve

Bioinformatics session with Vanessa Varaljay.

### Lab 2:

The end the week, the only process that needed to occur Friday was to check the final overnight OD of the induction test of pY71-bFMO. After this was completed, they took the rest of the day off.

# 7/22/2019 - 7/26/2019

## Monday

### Lab 1:

LabPats redid the 5μL standard curve because the wrong plate was
used for Friday's

The overnights for 3OC12 dose response curve were set up. The one
from 7/17 was not diluted properly so we needed to redo them.

### Lab 2:

To begin one of the last weeks in lab, the RX Lab students ran a 100 uL digest of chrP-bFMO and pSB3K3 in order to attempt to assemble the following day for the final construct. Additionally, they mini-prepped the BL21 DE3 chrP-bFMO and chrP-GFPa1 and a small digest was ran on them overnight for verification of band size. Finally, patches of both chrP constructs were made for both the RX and the RH labs.

## Tuesday

### Lab 1:

RH members went over to RX to work with running gels.LabPats
analyzed data. Only T7-mutated with and without Trp worked (turned
blue). No GFPa1 circuit fluorescence was detected. The
transformation of ChrO-bFMO (on a 20ng Kan plate) and ChrP-GFPa1
worked.A bFMO extraction was also done. 50μL of the overnight was
added to 750μL of DMSO.

mg/mL DMSO \* 750μL DMSO/50μL culture. Multiply the raw data by 15
and convert using the indigo standard curve (conversion factor
5.30E-5)

### Lab 2:

On this Tuesday, the RX Lab students started the day by running a gel of the digested pSB3k3 and the chrP-bFMO. The picture of the gel showed that neither of the plasmids were correctly cut. As a result, they started new overnight cultures of pSB3k3 and started a new overnight digest of chrP-bFMO cutting with Pst1, EcoR1, and both of them combined in order to find the issue. Additionally, they started a PCR of the pETlla-chrB that ran overnight. Finally they started overnight culture from patches of chrP-bFMO and chrP-GFPa1 for induction the following day.

## Wednesday

### Lab 1:

LabPats spent the day at UES working on wiki content, data analysis
consisting mostly of converting our readings to MEFL/particle, and
our presentation for Dr. Rajesh Naik. At 1:00pm RX members came
over to RH to get photos of the entire team doing labwork with the
base photographer. Then RH went back to UES to continue working.

### Lab 2:

Entering lab early in the middle of the week, the RX Lab Students began the induction of chrP-bFMO and chrP-GFPa1. They read the OD after induction at hours 1, 2, and 4. Additionally, they assembled and transformed the PCR of the pET11a-chrB from the previous day.

## Thursday

### Lab 1:

Cell free reactions w/bFMO were done.

Plate Setup was 25μL reactions in a 384 well plate B2-B4: T7
mutated bFMO + Trp

B5-B7: T7 mutated bFMO

B8-B10: ChrP bFMO + Trp

B11-B13 ChrP bFMO

B14-B16 DMSO only

Lyophilization of cell free reactions was also done.

### Lab 2:

As the summer starts to wind down, the RX Lab Students enter lab hoping for good results from their induction from the previous day. Sadly, no GFP was expressed in the induced culture and no indigo color was present, but the color was darker. Seeing these results, the RX Lab Students started new cultures of chrP-bFMO and chrP-GFPa1 and let them grow the entire day. Additionally, a gel was ran on the PCR performed the previous day and the results of the gel showed that the chrB was cloned. Another, larger PCR was setup overnight for extraction and assembly the following day. After the initial lab work was completed, the RX Lab Students headed to UES where they worked on the presentation for DR. Naik and wiki content. Following this work, they had a full team meeting where current progress and team goals and jobs were assessed. To finish the day, they headed back into lab where they induced the cultures of chrP-bFMO and chrP-GFPa1 while adding tryptophan to some of the chrP-bFMO cultures. IPTG was tested at 1 uM and 01. uM.

## Friday

### Lab 1:

Labpats rehydrated the reactions for Thursday.

A lot of time was spent talking with our mentor, Amy, about was to
complete medal criteria of measurement (bronze), validation
(silver) and improvement of an existing part (gold).

### Lab 2:

Coming into lab at the end of the week, the RX Lab Students were disappointed to see that the induced cultures of chrP- bFMO and chrP-GFPa1 did not change colors or fluoresce. New colonies of all of the constructs were started over the weekend. Finally, the students met up with the rest of the team to work on wiki content and the presentation for Dr. Naik.

# 7/29/2019 - 8/1/2019

## Monday

### Lab 1:

LB broth was made, The ChrP-mRFP was transformed and C4-HSL Dose
response curves were done and put in the plate reader(Amy glycerol
stock #7)

Overnights of ChrP-bFMO, chrP-GFPa1, ChrP-bFMO + Trp, pY71-bFMO,
and pY71-sfGFP were made and put in the incubator. LabPats tested
the paper sensor with fluorescein. After seeing success, cell free
tests on paper were run with pY71-sfGFP and bFMO (w/DNA, Trp,
indole made indole)

### Lab 2:

The RX Lab Students started off the week by heading into lab. They took the culture that Chia had grown up on Sunday and began a mini-prep of colonies of chrP-bFMO, chrP-GFPa1, pY71-bFMO, and C4-HSL-bFMO. Once the DNA was collected, they used the QuBit in order to discover the concentrations. The concentrations were found to be too low so they decided to grow up more overnight culture for another mini-prep the following day. Additionally they picked 4 colonies of each successful transformation of pET11a-chrB; PCR and G-Block. After the lab work was completed, they headed to UES to work on human practices and the presentation for DR. Naik which was to occur on Thursday, the 1st of August.

## Tuesday

### Lab 1:

Maxipreps

Cell free reaction

### Lab 2:

The RX Lab Students came in early to lab knowing a lot of lab work was ahead of them. They mini-prepped 10 mL of 4 colonies of chrP-bFMO, 4 colonies of chrP-GFPa1, 3 colonies of pY71-bFMO, and 3 colonies of C4-HSL-bFMO. They also mini-prepped 4 mL of 4 colonies of pET11a-chrB from PCR and 4 colonies of pET11a-chrB from G-Block. After all of the DNA was successfully collected, all of the DNA was QuBited for concentrations. Finally the DNA was sent out for sequencing. Additionally, all of the cultures were frozen down and stored in the -80 degree Celsius freezer. Once the lab work was completed, they headed to UES where they continued working on the presentation for Dr. Naik.

## Wednesday

### Lab 1:

One member of RH took the cell free data off the plate reader and
graphed it. Then the team met at UES to practice the presentation
for Dr. Naik.

### Lab 2:

Coming into lab in the middle of the week, the RX Lab Students split up in order to accomplish different objectives for the day. Two of the students went into lab in order to digest the pET11a-chrB from both transformations from PCR and G- Blocks. The two hour digest was ran on a gel and 7 out of the 8 transformations appeared to be successful. Additionally, all of the 8 colonies of pET11a-chrB were transformed into BL21 (DE3) and left to grow overnight. While the lab work was being completed, the other member of the RX Lab Students went to UES for the entire day in order to work on the presentation for DR. Naik the following day. Once the lab work was completed, the entire team met at UES in order to practice the presentation.

## Thursday

### Lab 1:

Presented to Dr. Naik

Ran cell free reactions in triplicates. One reaction had No DNA +
DMSO and the other had pY71-bFMO + DMSO Concluded DMSO had no
effect on the reaction

### Lab 2:

Entering lab towards the end of the week, the RX Lab Students and the rest of the team prepared for the presentation they were giving. They gave the presentation and receive feedback for future implementations and additions into the project from Dr. Naik. After the presentation, they went back to lab where they transformed 4 colonies of pET11a-chrB (2 from G- Block and 2 from PCR) into BL21 (DE3).

## Friday

### Lab 1:

Checked different wavelengths for pY71-bFMO absorbance measurements (470 nm vs 620 nm) using the DMSO testing from 8/1
and using ChrP-bFMO colonies in water and there was no difference in measurements. Realized that our T7 promoter had an illegal
XbaI site so we began a mutagenesis to remove it.
Made more 50 Kan plates, followed protocol
Plates Protocol-Kanamycin
Began performing single point mutagenesis to mutate an illegal XbaI cut site in between our T7 promoter and RBS region in our
pY71-sfGFP
QuikChange Site-Directed Mutagenesis
We also plated the blank on a non-resistance (NR) plate.
Made a 200 mL overnight of the PET11a-chrB Sample 1 Colony 1 to maxiprep (Ampicillin resistance)

### Lab 2:

This Friday was the "official" final day in lab for the RX Lab Students. Before out-processing, they picked colonies of pET11a-chrB and set them in the incubator to grow over the weekend. They spent the rest of their day setting up their work space for their return and out-processed from lab.

# 8/5/2019 - 8/9/2019

## Monday

### Lab 1:

Checked different wavelengths for pY71-bFMO absorbance measurements
(470 nm vs 620 nm) using the DMSO testing from 8/1 and using
ChrP-bFMO colonies in water and there was no difference in
measurements. Realized that our T7 promoter had an illegal XbaI
site so we began a mutagenesis to remove it.

Made more 50 Kan plates, followed protocol
![page19image41893248](blob:https://wordhtml.com/0283cb51-b105-42b9-a48d-298cd05c7c2d)

Began performing single point mutagenesis to mutate an illegal XbaI cut
site in between our T7 promoter and RBS region in our pY71-sfGFP

![page20image41791104](blob:https://wordhtml.com/7ef1c06d-6e9c-4ac5-b1a0-207c0ee87089)

We also plated the blank on a non-resistance (NR) plate.

Made a 200 mL overnight of the PET11a-chrB Sample 1 Colony 1 to
maxiprep (Ampicillin resistance)

### Lab 2: Week of Monday, 8/5 - Friday, 8/9

All throughout this week in lab, the RX lab students focused on verifying the production of pET11a-Chrb and RBS mutations of chrP-bFMO and chrP-GFPa1. 4 protein gels were run during this period after various levels of induction were tested. To much disappointment of the RX Lab Students, non of the protein gels showed clear results of the production of the repressor. Additionally, multiple mutation PCRs and protocols, and assemblies were attempted in order to create the successful mutation. Finally, clones of ChrP-GFPa1 and ChrP-bFMO were sent off for sequencing.

## Tuesday

### Lab 1:

Transformations were mostly unsuccessful. Blank plated on NR plate
only had a slight film. The 5 uL, 20 ng/μL reaction did have a few
colonies. So we made 7 overnights (6 colonies + 1 Blank) with 3 mL
of LB, and 3 μL of 50 ng Kanamycin. Maxiprep was unsuccessful
because there was some confusion over the last two steps that led
to a contaminated sample. So we redid a 200 mL overnight to repeat
the maxiprep again tomorrow. We concluded that the older DH5α cells
were used so we used newer DH5α cells to repeat the mutagenesis of
the 20 ng/μL reaction. We also began a mutagenesis for the T7
promoter in our pY71-bFMOplasmid using the same protocol with minor
revisions.

![page20image41515456](blob:https://wordhtml.com/dd5610bf-2d91-45d3-9510-95c41c03d8ca)

Master Mix for 7 reactions (4 pY71-bFMO, 1 pY71-sfGFP, 1 Blank, 1
extra)

5 iProof Buffer- 35μL

DNA

T7mut-F- 3.5μL (10x dilution of primers- 1 μL of each primer to 9
μL of water)

T7mut-R- 3.5μL

DNTP- 3.5μL

Water- 122.5 μL

iProof polymerase- 3.5μL

Total: 171.5/7=24.5 μL per reaction

pY71-sfGFP (20 ng/μL)

dilute 1 μL of DNA to 9 μL of water

Add 0.5 μL to corresponding reaction

pY71-bFMO - dilute 1 μL of DNA to 9 μL of water- 246.54 ng/μL is
about 250 ng/μL 5 ng/μL= 1:50 (49 μL) of water

10 ng/μL= 1:25 (24 μL) of water

20 ng/μL= 1:12.5 (11.5 μL) of water 50 ng/μL= 1: 5 (4 μL) of water

Add 0.5 μL of each diluted DNA sample to corresponding reactionPCR
Revisions:

For pY71-sfGFP: 68 °C for 75 seconds

For pY71-bFOMO 68 °C for 100 seconds

Left transformations overnight. Autoclaved some flasks, bottles,
and μL tubes.Sent pY71-sfGFP, pY71-bFMO, ChrP-GFPa1, ChrP-bFMO for
sequencing Primers (10x dilution needed):

seq\_pY71\_F

seq\_pY71\_R

pY71-sfGFP F- BGC225

pY71-sfGFP R- BGC226

pY71-bFMO F- BGC227

pY71-bFMO R- BGC228

ChrP-GFPa1 F- BGC229

ChrP-GFPa1 R- BGC230

ChrP-bFMO F- BGC231

ChrP-bFMO R- BGC232

## Wednesday

### Lab 1:

The maxi prepped sequences for PET11a-ChrB were confirmed.

Nanodrop for Maxiprep of Pet11A-ChrB

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | Concentration (ng/μL) | Ratio 260/280 |
| 2 | PET11a | 360.6 | 1.9 |
| 3 | EB | -0.7 | 0.87 |

LabPats mini prepped pY71-sfGFP (5μL, 20ng/μL) overnights using
miniprep kit. Glycerol stocks of this were made and labeled GS1,
GS2... GS6 sfGFP. They are in the iGEM 2017 box.T7-mut-pY71-sGFP

Miniprep Nanodrop Results

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | Concentration (ng/μL) | ratio 260/280 |
| 2 | EB #1 | -0.8 | 169.11 |
| 3 | EB #2 | -0.1 | 0.75 |
| 4 | T7-mut-sfGFP col. 1 | 143.0 | 1.88 |
| 5 | T7-mut-sfGFP col. 2 | 82.4 | 1.91 |
| 6 | T7-mut-sfGFP col. 3 | 83.8 | 1.90 |
| 7 | T7-mut-sfGFP col. 4 | 176.7 | 1.65 |
| 8 | T7-mut-sfGFP col. 5 | 89.3 | 1.90 |
| 9 | T7-mut-sfGFP col. 6 | 87.9 | 1.92 |
| 10 | EB #3 | 0.2 | 2.31 |

LabPats also did sequencing. The set-up was 8μL of DNA and 4μL of
10X primers.

T7 mut-sfGFP #1 F BGC233 R BGC 234

T7 mut-sfGFP #2 F BGC235 R BGC 236

T7 mut-sfGFP #3 F BGC237 R BGC 238

T7 mut-sfGFP #4 F BGC239 R BGC 240 (this sequence was confirmed!)

T7 mut-sfGFP #5 F BGC241 R BGC 242

T7 mut-sfGFP #6 F BGC243 R BGC 244

Finally, overnights of pY71-bFMO (T7-mut) were made with 3mL of LB
and 3μL of 50 Kan.

## Thursday

### Lab 1:

Glycerol stocks of T7 mut-pY71-bFMO were made. They are labeled as
GS bFMO 1... GS bFMO 10. 100μL of each overnight was put into a 60%
glycerol stock.Following the protocol from 8/7/2019, the overnights
were mini prepped and nano dropped. (ng, μL) in the segment #
column is for mutagenesis cone and transformation amount. For #1
and #9 DNA was diluted.

Nanodrop Results of Miniprepped Overnights

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | A | B | C | D |
| 1 | Segment # | Concentration (ng/μL) | Ratio 260/280 |  |
| 2 | EB #1 | 0.3 | 0.90 |  |
| 3 | BGC 245 T7- mut-bFMO #1(5ng, 1μL) | 173.5 | 1.75 | \*sequenced confirmed\* |
| 4 | BGC 246 T7- mut-bFMO #3(5ng, 5μL) | 156.1 | 1.86 |  |
| 5 | BGC 247 T7- mut-bFMO #2(5ng, 5μL) | 138.1 | 1.86 |  |
| 6 | BGC 248 T7- mut-bFMO #4(10ng, 1μL) | 130.6 | 1.86 |  |
| 7 | BGC 249 T7- mut-bFMO #5(10ng, 1μL) | 117.7 | 1.85 |  |
| 8 | BGC 250 T7- mut-bFMO #6(10ng, 5μL) | 128.6 | 1.85 |  |
| 9 | BGC 251 T7- mut-bFMO #7(10ng/5μL | 121.4 | 1.86 |  |
| 10 | BGC 252 T7- mut-bFMO #8(10ng/1μL) | 158.3 | 1.79 |  |
| 11 | BGC 253 T7- mut-bFMO #9(20ng, 5μL) | 187.4 | 1.80 |  |
| 12 | BGC 254 T7- mut-bFMO #10(20ng, 5μL) | 102.5 | 1.86 |  |
| 13 | BGC 255 T7- mut-sfGFP #2(20ng, 5μL) | 137.3 | 1.85 |  |
| 14 | EB #2 | 0.4 | 0.52 |  |

LB broth and a sleeve of 50Kan plates were also made.

## Friday

### Lab 1:

LabPats began making the indigo standard curve. 7.28mg of indigo
was put into 728μL of DMSO so there was a 0.01ng/μL concentration.
60μL of indigo was put into the 1st PCR tube. Each successive tube
had 30μL DMSO in them. 30μL of indigo from tube 1 was pipetted into
tube 2, 30μL of tube 2 was pipetted into tube 3, etc until liquid
became clear (approximately 16 tubes). This was run on the plate
reader at an absorbance at 620nm in a 384 well plate. The results
were inconsistent so the standard curve was repeated. The 17th well
in the plate was a blank of pure DMSO.

# 8/12/2019 - 8/16/2019

## Monday

### Lab 1:

An overnight of T7-mut-pY71-sfGFP #4 was made to maxiprep. It
consisted of 200mL of LB and 200μL of 50Kan. Overnights of
pY71-sfGFP and T7-mut-pY71-sfGFP #4 were also made for measurement.
These had 3mL of LB, 3μL of 50Kan, and were inoculated with G5
J7-mut-sfGFP #4 or pY71-sfGFP maxiprep.Received chrP-bFMO and
chrP-GFPa1 minipreps and NEB5 plate with potentially an RBS in it.
There were 4 minipreps each with an approximately 200ng/μL
concentration for each. These were not nanodropped. Primers
bFMO425-443, bFMO932- 950, and C4-HSL-bFMO seg Rev were received.
Sequencing set-up was 8μL of DNA and 4μL of 10X primers.

pY71-bFMO (redo) BGC256

ChrP-bFMO #1 BGC257 (443 primer)

ChrP-bFMO #1 BGC258 (950 primer)

ChrP-bFMO #1 BGC259 (F seg-pY71- F primer)

ChrP-bFMO #2 BGC260 (443)

ChrP-bFMO #2 BGC261 (950)

ChrP-bFMO #2 BGC262 (F)

ChrP-bFMO #3 BGC263 (443)

ChrP-bFMO #3 BGC264 (950)

ChrP-bFMO #3 BGC265 (F)

ChrP-bFMO #4 BGC266 (443)

ChrP-bFMO #4 BGC267 (950)

ChrP-bFMO #4 BGC268 (F)

ChrP-GFPa1 #1 BGC269 (F)

ChrP-GFPa1 #2 BGC270 (F)

ChrP-GFPa1 #3 BGC271 (F)

ChrP-GFPa1 #4 BGC272 (F)

Also sequenced 3OC12 and RhlR circuits (10X dilution of maxipreps)

pLas-sfGFP- BGC273- AME1012

pLas-sfGFP- BGC274- AME1013

RhlR- BGC275- AME1012

RhlR- BGC276- AME1013

Made 4 overnights in triplicates of pY71-bFMO (NEB5) from a
glycerol stock and plate from RX to check for mutation because
there was a point mutation in our pY71-bFMO that caused an amino
acid change. The overnights consisted of 3mL of LB and 3μL of
50Kan. 3 overnights were from the pY71-bFMO glycerol stock #1 and 9
overnights were made from the plate (3 from sample 1, 3 from sample
2, and 3 from sample 3).

### Lab 2: Week of Monday, 8/12 - Friday, 8,16

On the final week of lab, the RX Lab Students focused on the same processes as the previous weeks since the sequencing showed that the mutation had not worked. All the same protocols were followed until the end of time inside of lab.

## Tuesday

### Lab 1:

Overnights of the pY71-sfGFP did not grow anything so LabPats redid
them using a colony from a 9X pY71-sfGFP (DH5∝) plate from 7/22/19.
20μL of LB + colony meant that each overnight was 5μL. 4 overnights
of T7-mut-sfGFP #4 were made from glycerol stock #4 (also in
DH5∝)T7-mut-sfGFP #4 was maxiprepped.

Nanodrop Results of Maxiprepped T7-mut-sfGFP #4

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | Concentration (ng/μL) | Ratio 260/280 |
| 2 | EB | 0.0 | -0.18 |
| 3 | T7-mut-sfGFP #4 | 690.2 | 1.90 |
| 4 | EB #2 | -0.3 | 2.92 |

5μL Cell free reactions of pY71-sfGFP and T7-mut-sfGFP were set up
(25μL aliquoted into 5μL reactions). The plate reader was set up to
read fluorescence 480/510 at sensitivity 19 and 30O.C. The plate
was a 96 well V-bottomed plate with a membrane.

(2X dilution of Maxiprep 5 μL water and 5μL DNA)

No DNA (N)

Wells B2-B5

10μL Premix

9μL Extract

6μL Water

Blank (Water)

Wells C6-C9

pY71-sfGFP (P)

189ng/μL

Wells B6-B9

10μL Premix

9μL Extract

3μL DNA

3μL Water

T7-mut-sfGFP (T)

Wells C2-C5

10μL Premix

9μL Extract

1.6μL DNA

4.4μL Water

25μL lyophilization reactions were done. They were same cell free
reactions as the ones run through the plate reader with 3reactions
of No DNA, 3 reactions of pY71-sfGFP (P), and 3 reactions
T7-mut-sfGFP (T).25μL cell free reactions aliquoted into 2.5μL
reactions were also run on the paper-based sensor. There were two
25μL reactions of No DNA, one of pY71-sfGFP and one of
T7-mut-sfGFP. There were 2 coupons each divided into 15 areas with
500μL of water. The coupon with the green sticker was for
pY71-sfGFP, and the other with a purple sticker was for T7-mut-
sfGFP. A 2.5μL reaction was pipetted into each of the 15 areas as
shown below.

Made overnights of BL21-DE3 glycerol stocks (B1-B4) from 7/12/19.
Miniprepped pY71-bFMO (NEB5) overnights, but only the first
triplicate of each.

Nanodrop Results of pY71-bFMO (1st Triplicate Only)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | A | B | C | D |
| 1 |  | Concentration (ng/μL) | Ratio 260/280 | Sequence # |
| 2 | EB #1 | 0.0 | 0.16 |  |
| 3 | pY71-bFMO Glycerol Stock #1 | 38.0 | 1.97 | BGC 277 |
| 4 | pY71-bFMO Sample 1Triplicate 1 | 119.5 | 1.87 | \*\*BGC 278 |
| 5 | pY71-bFMO Sample 2Triplicate 1 | 28.2 | 1.89 | BGC 279 |
| 6 | pY71-bFMO Sample 3Triplicate 1 | 94.0 | 1.73 | BGC 280 |
| 7 | EB #2 | 0.2 | -1.25 |  |

\*\*BGC 278 sequence was confirmed as the correct bFMO sequence.

## Wednesday

### Lab 1:

T7-mutated-pY71-sfGFP overnights grew. 100μL of each overnight were
measured in the plate reader (blanks of LB only were included). The
settings included fluorescence 480/510, and sensitivity at 6 which
is the default for the plate reader.

The overnights of pY71-bFMO BL21-DE3 were mini prepped to check for
mutation (all glycerol stock B1-B4 had the mutation). B1-B3 had a
pink pellet and B4 had a blue pellet.

Nanodrop Results of pY71-bFMO BL21-DE3 (B1-B4)

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | A | B | C | D |
| 1 |  | Concentration (ng/μL) | ratio 260/280 | Segment # (F primer only) |
| 2 | EB #1 | -0.8 | 2.04 |  |
| 3 | B1 | 168.3 | 1.91 | BGC 281 |
| 4 | B2 | 177.6 | 1.91 | BGC 282 |
| 5 | B3 | 164.0 | 1.91 | BGC 283 |
| 6 | B4 | 189.0 | 1.91 | BGC 284 |
| 7 | EB #2 | -0.1 | -31.27 |  |

The indigo standard curve was remade with 4.2mg into 420μL of DMSO.
The 1st tube had 60μL of indigo. The 2nd, 3rd, 4th, etc. tube had
30μL of DMSO. 30μL of indigo from tube 1 was put into tube 2. 30μL
of tube 2 was put in tube 3, etc until liquid became clear. Each
was the put into the plate from B2-B17, C2-C17, and D2-D17 (C and D
are triplicates) with read absorbance on the plate reader at
620nm.LabPats made a fluorescein 25μL standard curve. First a 10x
dilution of 100μM stock of fluorescein needed to be done. 20μL
stock into 180μL of 1X PBS were put into tube 1. The same dilution
procedure as the indigo standard curve was used for this dilution.
Each of the following tubes (2-6) begins with 100μL of 1X PBS and
tube 7 has 100μL of 1X PBS only. 25μL of each tube was put into a
well (tube one corresponds with well B2) in a clear v-bottom 96
well plate. The setup was B2-B8, C2-C8 and D2-D8 (C and D are
triplicates). 2 reads of the plate were done one at sensitivity 19
and the other at sensitivity 6 (default). Both were read at
480/510.The pY71-sfGFP and T7-mut-pY71-sfGFP were lyophilized with
25μL water. They were then read in a 384 well plate. Wells B2- B4
had T7-mut-pY71-sfGFP, wells C2-C4 had pY71-sfGFP, and wells D2-D4
had no DNA. The fluorescence reading was 480-510. However the
purple adapter (for absorbance) was left in so they must be
redone.The overnights from 8/13 were read for absorbance as well.
They were put in a 96-well plate with a flat, clear bottom. Needed
a 2X dilution of the overnights (50μL of overnight and 50μL of LB).
Two plates were run: one at an absorbance of 600nm and the other at
an absorbance of 620nm. Only the pY71-sfGFP ones were read so this
too needs to be redone. Glycerol stocks were made for the
pY71-sfGFP overnights. They are being stored in the iGEM 2019 box
labeled not recommended for use. LabPats also made a 200mL
overnight of pY71-bFMO GnSn NEBS Sam1 Trp1 to maxiprep. It
consisted of 200mL LB and 200μL of 50 Kan.

## Thursday

### Lab 1:

LabPats maxi prepped pY71-bFMO G.S. NEB5 Sample 1 Triplicate 1. It
is currently being stored in lambda box.

Nanodrop Results of pY71-bFMO Maxiprep

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | concentration (ng/μL) | ratio 260/280 |
| 2 | EB #1 | 0.0 | 0.17 |
| 3 | pY71-bFMO Sam1 Trp1 | 148.3 | 1.9 |
| 4 | EB #2 | -0.2 | -0.55 |

A mutagenesis was performed on the pY71-bFMO G.S. NEB5 Sample 1
Triplicate. 1, however it needs to be redone because the wrong
primers were used.The 25μL fluorescence standard curve, and the
lyophilization of T7-mut-pY71-sfGFP and pY71-sfGFP from the 14th
were redone. The standard curve was done in a 384 well plate.
Overnights consisting of 3mL of LB, 3μL of 50 Kan, and pY71-sfGFP
glycerol stock #4. and overnights with T7-mut-pY71-sfGFP glycerol
stock #4 were made.

## Friday

### Lab 1:

Measured overnights of pY71-sfGFP + T7mut-pY71-sfGFP. A 2X dilution
was done to fit the curve with 50 LB and 50 of the overnight. Both
fluorescence and absorbance was measured with the plate reader
settings at 480/510 for fluorescence and abs. at 600 nm. The wells
used were B2-B4 for pY71-sfGFP, B5-B7 for T7mut-pY71-sfGFP, and
B8-B10 for Blanks with just LB.Rehydrated the lyophilization of
pY71-sfGFP and T7mut-sfGFP with 25 μL of water. A 384 well plate
was used. Bubbling occurred in some of the wells. B2-B4 had No DNA,
B5-B7 had pY71-sfGFP, and B8-B10 had T7mut-pY71-sfGFP. Triplicate 2
was placed in C8 well instead of B8 so an endpoint read was done on
that well.LabPats also performed mutagenesis of pY71-bFMO to
correct the T7 promoter.

Master Mix

5 iProof Buffer- 30μL

DNA

T7mut-F- 3μL

T7mut-R- 3μL

DNT p- 3μL

H2O- 105 μL

iProof polymerase- 3μL

10x dilution of primers- 1 μL of each primer- 9 μL of water

pY71-bFMO Sample 1: Triplicate 1- 119.5 ng/μL is about 120 ng/μL

5 ng/μL= 1:24 (23 μL) of water

10 ng/μL= 1:12 (11 μL) of water

20 ng/μL= 1:6 (5 μL) of water

50 ng/μL= 1: 2.4 (1.4 μL) of water

Transformed and left on plates on bench over the weekend. Also
transformed pY71-bFMo into BL21 cells, but needs to beredone because it
was meant to be BL21-DE3 cells.

# 8/19/2019 - 8/23/2019

## Monday

### Lab 1:

Blanks and pY71-bFMO BL21 plates grew, but the rest did not. They were
put into the 37°C incubator for a couple of hours. pY71-bFMO (sample 1
triplicate 1) was transformed into BL21-DE3 cells, plated and put in
incubator overnight.Minipreps #2 that was sequenced on 8/12/19 was
nanodropped and used in the ChrP bFMO and ChrP-GFPa1 RBS mutagenesis.

Nanodrop Results of ChrP-GFPa1 and ChrP-bFMO

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | Concentration (ng/μL) | 260/280 |
| 2 | EB #1 | -0.8 | 1.22 |
| 3 | ChrP-GFPa1 | 156.3 | 1.90 |
| 4 | ChrP-bFMO | 311.4 | 1.90 |
| 5 | EB #2 | -0.7 | 0.94 |

Mixes

2-10 ng DNA

1 μM primer A- 0.5 μL

1 μM primer B- 0.5 μL

200 μM DNTPs- 0.5 μL

3U enzyme (iProof polymerase)- 0.5μL Buffer (HF)- 10 μL

Water- To 50 μL total

DNA in 5ng, 10ng, 20ng GFPa1

5 ng- 1:31.2 (30.2 μL water)

10 ng- 1:15.6 (14.6 μL water)

20 ng- 1: 2.8 (6.8 μL water)

Master Mix (n=4)

GFPa1

RBS\_F\_GFPa1... 2 μL

RBS\_R... 2 μL

DNTP... 2 μL

Polymerase... 2 μL

Buffer... 40 μL

Water... 148 μL (196 μL/4)= 49 μL

bFMO

5 ng- 1:62.2 (61.2 μL water)

10 ng- 1:31.3 (30.1 μL water)

20 ng- 15.55 (14.55 μL water)

bFMO

RBS\_F\_bFMO... 2 μL

RBS\_R... 2 μL

DNTP... 2 μL

Polymerase... 2 μL

Buffer... 40 μL

Water... 148 μL (196 μL/4)= 49 μL

Added 1 μL of DNA to each reaction except blank PCR cycle GFPa1

1 through 3- 12x

1. 95°C 5 min
2. 95°C 5 min
3. 60°C 1 min
4. 72°C 75 sec
5. 46°C 1 min
6. 72°C 7.5 min

1 through 3- 12x

1. 95°C 5 min
2. 95°C 1 min
3. 58°C 1 min
4. 72°C 100 sec
5. 46°C 1 min
6. 72°C 7.5 min

Added 0.5 μL of DpNI, incubated at 37°C for 2 hours,

Made overnights of 3OC12 circuit (PLas\_sfGFP from JD #5) to take
pictures of. Three overnights had 3OC12 and consisted of 3μL of 3OC12 +
3 μL chloramphenicol. Three other overnights had 3μL of DMSO + 3 μL of
chloramphenicol.

Colonies of T7mut-bFMO grew in plates. LabPats used 20 ng, 5 μL plate.
2 colonies were made into 200 mL overnights to maxiprep, and 4 colonies
were made into 3 μL overnights to miniprep. Plates that weren't used
were left in the incubator overnight.

## Tuesday

### Lab 1:

pLas- sfGFP overnights grew and T7mut-bFMO maxiprep and miniprep
overnights grew. LabPats began transforming ChrP- GFPa1 and ChrP-bFMO
RBS MUT Genes

1.) 100 μL of DH5∝

2 μL DNA20 uL 5x KCM

78 uL H2O (Nuclease free)

2.) Ice 30 minutes

3.) Heat shock 45 seconds @ 42°C

4.) Ice 2 minutes

5.) + 900 uL LB

6.) Incubate for 30 minutes

7.) Spin at 800 RPM for 5 minutes

8.) Took 900 uL out

9.) Resuspend in 200 uL

10.) Plate 100 uL and put in incubator @ 37°CMaxiprepped
T7-mut-pY71-bFMO colony 5 and 6. Forgot to make immediate glycerol
stocks so three overnights were made using the spun down pellet. Two
overnights had 3mL LB, 3uL Kan, and the pellet. There was one blank
with LB.

and stored PCR in 4°C fridge.

Nanodrop Results of Miniprepped T7-mut-bFMO Colonies 5 and 6

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | Concentration (ng/uL) | 260/280 |
| 2 | EB #1 | -0.3 | -8.18 |
| 3 | T7 mut-pY71- bFMO col. 5 | 576.2 | 1.89 |
| 4 | T7 mut-pY71- bFMO col. 6 | 877.5 | 1.89 |
| 5 | EB #2 | -0.6 | 1.01 |

Miniprepped T7-mut-bFMO Colony 1-4 Overnights. Followed Protocol
from 7/17/19. They are labeled MP1 T7mut-bFMO 8/20/19... MP4
T7mut-bFMO 8/20/19 and are in Lambda box.

Nanodrop Results of Miniprepped T7-mut-bFMO Colonies 1-4

|  |  |  |  |
| --- | --- | --- | --- |
|  | A | B | C |
| 1 |  | Concentration (ng/uL) | 260/280 |
| 2 | EB #1 | 1.1 | 0.93 |
| 3 | T7mut- pY71- bFMO Col 1 | 157.9 | 1.87 |
| 4 | T7mut- pY71- bFMO Col 2 | 150.8 | 1.88 |
| 5 | T7mut- pY71- bFMO Col 3 | 150.2 | 1.88 |
| 6 | T7mut- pY71- bFMO Col 4 | 102.7 | 1.89 |
| 7 | EB #2 | 0.4 | 0.48 |

Sequencing Results

Miniprep 1- BGC285

Miniprep 2- BGC286

Miniprep 3- BGC287

Miniprep 4- BKC605

Miniprep 5- BGC606 Sequencing Confirmed

Miniprep 6- BGC607 Sequencing Confirmed

10x dilution of maxipreps: 1uL of maxiprep, 9uL H2O, F Primer only
(because it’s a 10x dilution), 2.5uL P and 22.5 uL H2O.

## Wednesday

### Lab 1:

25uL cell free reactions (T7mut-bFMO Colony 5) Indole- 0.0085 g to
1.7 mL DMSO is about 5mg/uL

![page32image42002368](blob:https://wordhtml.com/3da413ec-6669-42b0-a827-4be9721de06f)

A. No DNA (run in triplicates)

10 uL Premix

9 uL Extract

6 uL Water

B. DNA+Trp

10 uL Premix

9 uL Extract

2.5uL Trp

1uL DNA

2.5 uL Water

C. DNA + Indole

10 uL Premix

9 uL Extract

2.5uL indole

1uL DNA

2.5 uL Water

D. DNA + DMSO

10uL Premix

9uL Extract

1uL DNA

0.5uL DMSO

4.5uL Water

E. Trp

10uL Premix

9uL Extract

2.5uL Trp

3.5uL Water

F. Indole

10uL Premix

9uL Extract

2.5uL Indole

3.5uL Water

Used Wells I2-I23. I1 and I24 were blank (had nothing in them).

Transformed T7mut-bFMO maxiprep colony 5 into BL21-DE3 and set up
overnights of ChrP-bFMO and ChrP-GFPa1.

Sequencing

ChrP-GFPa1 #1BKC608

ChrP-bFMO #1BKC609

## Thursday

### Lab 1:

LabPats worked on measurement write up. The sequencing results came back,
and it seems that colonies were switched. 4 colonies of GFPa1 grew and 1
colony of bFMO. The RBS was successfully put into ChrP-GFPa1 which was
originally labeled as ChrP-bFMO #1. The RBS was missing the first “A” in
ChrP-bFMO which was originally labeled ChrP-GFPa1 #1

Transformed ChrP-GFPa1 (sequencing confirmed) and ChrP-RBS-bFMO
into BL21-DE3 cells

Cell free reactions from 8/21/19 of T7mut-bFMO at different
conditions did not work. DNA + indole and indole reactions were
high. It was realized that there was no DNA only condition so a DNA
titration was set up.

DNA Titrations

No DNA

10μL Premix

9μL Extract

6μL Water

5.13μL Water

DNA (20 ng/μl)

10μL Premix

9μL Extract

0.87μL DNA

4.3μL Water

DNA (40 ng/μl)

10μL Premix

9μL Extract

1.7μL DNA

DNA (60 ng/μl)

10μL Premix

9μL Extract

2.6μL DNA

3.4μL Water

DNA (80 ng/μl)

10μL Premix

9μL Extract

2.6μL DNA

2.5μL Water

Set up overnights of T7-ChrP-GFPa1 and T7-ChrP-bFMO from plates

A: T7-ChrP-GFPa- 17-22-19- col.1BL21 (DE3)

B: T7-ChrP-bFMO- 7-22-19- col.1BL21 (DE3)

C: T7-ChrP-RBS GFPa1- 8-20-19- col.1DH5∝

D: T7-RBS-bFMO- 8-20-19 DH5∝

E: T7-ChrP-GFPa1- 8-9-19- col.1NEB5∝

F: T7-ChrP-bFMO- 8-9-19- col.1NEB5∝

G: T7-mut-bFMO- 8-21-19- BL21 (DE3)

Table of What Makes Up Each Reaction

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | A | B | C | D | E | F | G |
| 1 |  |  | Media | Kan | Trp | IPTG | Colony (dissolved in 25 ul LB) |
| 2 | A | 3x | 3 mL | 3 |  |  | 2 |
| 3 | B | 3x | 3 mL | 3 |  |  | 2 |
| 4 |  | 3x IPTG | 3 mL | 3 |  | 1.5 | 2 |
| 5 |  | 3x Trp | 2.4 mL | 3 | 600 |  | 2 |
| 6 |  | 3x IPTG +TRP | 2.4 mL | 3 | 600 | 1.5 | 2 |
| 7 | C | 3x | 3 mL | 3 |  |  | 2 |
| 8 | D | 3x | 3 mL | 3 |  |  | 2 |
| 9 |  | 3x Trp | 2.4 mL | 3 | 600 |  | 2 |
| 10 | E | 3x | 3 mL | 3 |  |  | 2 |
| 11 | F | 3x | 3 mL | 3 |  |  | 2 |
| 12 |  | 3x Trp | 2.4 mL | 3 | 600 |  | 2 |
| 13 | G | 3x | 3 mL | 3 |  |  | 2 |
| 14 |  | 3x IPTG | 3 mL | 3 |  | 1.5 | 2 |
| 15 |  | 3x Trp | 2.4 mL | 3 | 600 |  | 2 |
| 16 |  | 3x IPTG + Trp | 2.4 mL | 3 | 600 | 1.5 | 2 |

## Friday

### Lab 1:

LabPats continued data analysis including on graphpad. Looking at
results, only T7mut with and without the Trp worked (turned blue).
No GFPa1 circuit showed fluorescence. The transformation of
ChrP-bFMO and ChrP-GFPa1 worked. The ChrP- bFMO was grown on a 20ng
Kan plate.bFMO extraction

50 μL of overnights into 750 μL of DMSO (μg/(μL DMSO))\*(750μ L
DMSO/50 μL culture) Multiply raw data by 15 (because 750/50)

Convert using the standard curve for indigo where 5.30E-5
conversion factor for indigo.

Plate Setup for bFMO absorbance measurement. (384 well plate with
25μl wells)

B2-B4- T7mut bFMO + Trp

B5-B7- T7 mut bFMO

B8-B10- Chrp bFMO +Trp

B11-B13 Chrp bFMO

B14-B16 DMSO only

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# Team:US AFRL CarrollHS/Experiments

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Experiments\_Header\_Final

# Protocols

# Transformations

# **Bl21**

1. Thaw 50 µL vial of BL21 cells on ice
2. Inoculate 2µL DNA into 50µL BL21 cells
   * Incubate cells on ice for 30 minutes
   * Heat shock at 42℃ for 10 seconds
   * Incubate on ice for 5 minutes
3. Inoculate 250µL LB into vial
4. Plate 1x/9x

# **Gel protocols**

(1% Agarose Electrophoresis Gel)

1. Add 0.35 g agarose to 35 mL 1x TAE in 100mL conical flask
2. Heat in microwave for 35 seconds
3. Swirl and heat in microwave for 25 seconds
4. Swirl and heat in microwave for 15 seconds
5. Swirl and heat in microwave for 7 seconds
6. Swirl and ensure no ‘floaties’
7. Cool outside of conical flask with water until ‘hand not’
8. Pour into gel mold and add well comb

# **Gel Extraction**

1. Use ethanol-wiped scalpel to excise appropriate bands
2. Mix and incubate at 50°C for 10 min
   * Vortex every 2 mins until gel slice is completely dissolved
3. 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
4. Add 700 mL Buffer NT3
   * Centrifuge at 11,000 xg for 1 minute
   * Discard throughflow and place column back in same tube
5. Repeat step 4
6. 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
7. 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

# **Protein Gel Electrophoresis**

Sample Preparation

1. Prepare suitable amount of sample buffer (25 µl per reaction).
2. Determine protein concentration with NanoDrop.
3. Calculate required dilutions needed to get 150 µg protein in 75 µl of solution.
4. In the fume hood, add 25 µl of the sample buffer (blue) to 75 µl of protein samples (total volume of 100 µl). Centrifuge samples at 13000 rpm for 60 seconds.
5. Incubate samples for 5 minutes in a dry heating block at 90 ◦C.
6. Centrifuge samples at 13000 rpm for 60 sec.
7. Store all samples at −20 ◦C

Gel Preparation (Separating Gel)

1. Prepare the gel casts in holders. Fill the holder with water to check for leakage after 5 min. After leakage check, pour all water from gel casts and dry residual with paper towel.
2. To prepare separation-gel, ix the reagents listed in the table below with the proper volumes to receive a gel with desired composition.
3. Add TEMED and APS last, solidification of the gel will occur when these reagents are added.
4. Pipette gel mixture into gel casts up to a height approximately 1 cm below the gels comb.
5. Fill the remaining cast with isopropanol (if not available use distilled water).
6. Allow to harden for 30-60 min.
7. Pour all isopropanol (or water) from gel casts and dry residual with paper towel.
   * Ammonium Persulfate (APS) - 20 mg/ml
   * Acrylamide 30 % solution
   * TEMED
   * Separating Gel Buffer - 1.5 M Tris-HCl pH 8.8 with 0.4% SDS. Stored at 4 ◦C.
   * Stacking Gel Buffer - 0.5 M Tris-HCl pH 6.8 with 0.4% SDS. Stored at 4 ◦C

Gel Preparation (Stacking Gel)

1. To prepare stacking-gel, in a tube mix the reagents listed in table 4 below with the proper volumes to receive a gel with desired composition.
2. Add TEMED and APS last, solidification of the gel will occur when these reagents are added.
3. Pipette gel mixture on top of separating gel to fill cast and remove all air bubbles.
4. Fill the remaining cast with isopropanol (if not available use distilled water).
5. Insert the comb whilst the gel is still in liquid form. The comb will form wells to load samples when the gel turns solid.
6. Allow to harden for 45-60 min.

Running the gel

1. Mount the gels into the tank, remove combs and fill the inner chamber of the tank with 1x running buffer to the top. Fill a third of the volume in the outer chamber of the tank with 1x running buffer
2. Pipette 2 µl ladder and 10 µl sample into each well.
3. Connect power pack (red-red, black-black) and run at 100 V, 60 min or until the blue line almost reaches the bottom of the gel.
4. Once electrophoresis has finished, remove gels from the glass plates.
5. Carefully wash all equipment used for electrophoresis with water.

# **Plates**

# **Chloramphenicol**

1. Follow LB Agar Protocol
2. Put in water bath 42°C
   * Cool til not burning hand
3. Put on stirring plate
4. Put sterilized plates in Lamenia shield
5. Using micropipette put .5 mL of chloramphenicol into agar solution
   * 1000x dilution
6. Pour solution into plates

# **Kanamycin**

1. Follow LB Agar Protocol
2. Put in water bath 42°C
   * Cool til not burning hand
3. Put on stirring plate
4. Put sterilized plates in Lamenia shield
5. Using micropipette put .5 mL of 50 ng kanamycin into agar solution
   * 1000x dilution
6. Pour solution into plates

# **QIA Prep Spin Mini Prep Kit**

1. Example for 18 mL LB add
   * 18 L20 mg/ L Kanamycin (antibiotic)
   * 18 L 50 mg/ L ampicillin
2. Aliquot 3.5 mL into labelled round bottom falcon tubes
3. Using sterile toothpicks- pick a single colony forming unit (cfu) and inoculate tube
   * Incubate overnight at 37°C with 215 rpm shaking
4. To 100 mL 60% glycerol stock, add 300mL overnight culture to appropriately labelled tube
   * Store at -80℃
5. Pellet remaining cells by centrifugation in 1.5 mL tube at 13,2000 rpm for 1 min
   * Discard supernatant and repeat
6. 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
7. Centrifuge at 13, 200 rpm for 10 minute
8. Pipette supernatant into labelled QIA prep spin column
   * Centrifuge at 13, 200 rpm for 1 minute
   * Discard Flowthrough
9. Wash column by adding .5 mL Buffer PB \*regular for low copy number plasmids\*
   * Centrifuge at 13, 200 rpm for 1 minute
   * Discard flowthrough
10. Wash by adding .75 mL Buffer PE
    * Centrifuge at 13, 200 rpm for 1 minute
    * Discard flowthrow
    * Centrifuge for an addition minute to ensure removal of residual wash buffer
11. 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℃

# **ZYMO Pure II Plasmid Maxiprep Kit**

1. Take 1 1.5 ml microcentrifuge tube and place 200 microliters of ZymoPURE Elution Buffer (EB)
2. Place EB tube in a 50 C heat block
3. Using a 50 ml pipette tip, pipette 50 ml of your 200 ml overnight into 4 Falcon tubes
4. Centrifuge the Falcon tubes at 2,500 x g for 20 minutes to pellet the cells. Discard the supernatant.
5. Using a 25 ml pipette tip, pipette 14 ml of ZymoPURE P1 (red) to one of the Falcon tubes and resuspend the cells.
6. Transfer the 14 ml of your cells and P1 into another Falcon tube and resuspend. Repeat for each Falcon tube.
7. Add 14 ml of ZymoPURE P2 (green) and immediately mix by gently inverting tube 6 times. DO NOT VORTEX. Let it sit at room temperature for 2.5 minutes. Cells will be completely lysed when the solution appears clear, purple and viscous.
8. Add 14 ml of ZymoPURE P3 (yellow) and mix gently but thoroughly by inversion. DO NOT VORTEX. The sample will yellow when the neutralization is complete amd a yellowish precipitate will form.
9. Ensure the plug is attached to the Luer Lock at the bottom of the ZymoPURE Syringe Filter. Place the syringe filter upright in a tube rack and load the lysate into the ZymoPURE Syringe Filter and wait 5-8 minutes for the precipitate to float to the top.
10. Remove the Luer Lock plug from the bottom of the syringe and place the syringe into a clean 50 ml conical tube. Place the plunger in the syringe and push the solution through the ZymoPURE Syringe Filter in one continuous motion until approximately 33-35 ml of cleared lysate is recovered. Save the cleared lysate!
11. Add 14 ml of ZymoPURE Binding Buffer to the cleared lysate from step 5 and mix thoroughly by inverting the capped tube 10 times
12. Follow Vacuum Protocol below. Vacuum pump should be able to apply at least 400 mm Hg pressure
13. Ensure the connections of the Zymo-Spin V-P Column Assembly are finger-tight and place onto a vacuum manifold.
14. With the vacuum off, add the entire mixture from step 6 into the Zymo-Spin V-P Column Assembly, and then turn on the vacuum until all of the liquid has passed completely through the column.
15. With the vacuum OFF, add 5 ml of ZymoPURE Wash 1 to the 15 ml Conical Reservoir. Turn on the vacuum until all of the liquid has passes completely through the column.
16. With the vacuum OFF, add 5 ml of ZymoPURE Wash 2 to the 15 ml Conical Reservoir. Turn on the vacuum until all of the liquid has passes completely through the column.
17. Repeat Step 14.
18. Remove and discard the 15 ml Conical Reservoir and place the Zymo-Spin V-P Column in a Collection Tube. Centrifuge at 13,400 x g for 1 minute, in a microcentrifuge, to remove any residual wash buffer.
19. Transfer the column into a clean 1.5 ml microcentrifuge tube and add 200 microliters of the heated EB directly into the column matrix. Wait 10 minutes and then centrifuge at 13,400 x g for 1 minute.
20. Nanodrop to measure DNA concentration of your maxiprep.

# **QuikChange Site-Directed Mutagenesis**

1. Set up the series of various concentrations of dsDNA template (5, 10, 20, and 50 ng of dsDNA)
   * pY71-sfGFP ≈ 200 ng/μL

     5 ng/μL = 1:40 ( 39 μL of ddH₂O + 1 μL of pY71-sfGFP)

     10 ng/μL = 1:20 (19 μL of ddH₂O + 1 μL of pY71-sfGFP)

     20 ng/μL = 1:10 (9 μL of ddH₂O + 1 μL of pY71-sfGFP)

     50 ng/μL = 1:4 (3 μL of ddH₂O + 1 μL of pY71-sfGFP)
2. Did a 10x dilution of the primer #1 T7-mutF and primer #2 T7-mutR
3. Set up a master mix for 6 reactions ( 4 different concentrations of dsDNA, 1 blank, and 1 extra)
   * 30 μL of 5x iProof Buffer

     3 μL of T7-mutF

     3 μL of T7-mutR

     3 μL of DNTP

     105 μL of ddH₂O

     3 μL of iProof DNA polymerase
4. Place 24.5 μL of the master mix into 5 different labeled PCR tubes(5, 10, 20, 50 ng of pY71-sfGFP and a blank)
   * Add 0.5 μL of the corresponding DNA to each DNA tube

     Add 0.5 μL of ddH₂O to the blank labeled tube
5. PCR Protocol

Preheat lid to 98℃

|  |  |  |  |
| --- | --- | --- | --- |
| Segment | Cycles | Temperature (℃) | Time |
| 1 | 1 | 95 | 30 seconds |
| 2 | 12 | 95 | 30 seconds |
| 55 | 1 minute |
| 68 | 1 min/kb of plasmid length, 2.5 min for pY71-sfGFP |
| 3 | 1 | 4 | 2 minutes |

MAKE SURE TO REPEAT SEGMENT 2 FOR 12 CYCLES

6. After PCR reaction is finished, add 0.5 μL of Dpn I restriction enzyme.

Gently mix by pipetting the solution up and down several times. Spin down the reactions in a microcentrifuge for 1 minute

7. Immediately incubate reactions at 37 ℃ for 1 hour to digest

Can be done in either an incubator or a thermocycler

8. After incubation, do 2 plasmid transformations into DH5α cells for each PCR tube, 1 with 1 μL of DNA and the other with 5 uL of DNA

100 μL of DH5α cells

* Supplement the change of DNA used with ddH₂O:
  + If adding 1 μL of DNA

    Add 20 μL 5x KCM and 79 μL of ddH₂O,

    If adding 5 μL of DNA

    Add 20 μL 5X KCM and 75 μL of ddH₂O

* Ice for 30 minutes
* Heatshock for 45 seconds at 42 ℃
* Ice again for 2 minutes
* Add 900 μL of LB Broth
* Shake and incubate for an hour at 37 ℃
* Spin tubes down at 800 x g for 5 minutes
* Remove 1000 μL, and resuspend pellet
* Plate the whole tube

# **Gibson Assembly® Protocol (E5510)**

Set up the following reaction on ice:

1. Reaction volumes: *Use this table to calculate reaction volumes and set up the reaction. Remember to* ***input your total DNA fragment volume in cells B3 and C3*** *for assemblies with 2-3 fragments and 4-6 fragments, respectively.*

*- NEB recommends a total of* ***0.02–0.5 pmols of DNA fragments when 1 or 2 fragments*** *are being assembled into a vector and* ***0.2–1.0 pmoles of DNA fragments when 4–6 fragments*** *are being assembled. Efficiency of assembly decreases as the number or length of fragments increases. To calculate the number of pmols of each fragment for optimal assembly, based on fragment length and weight, we recommend using NEB's online tool,*[*NEBioCalculator*](http://nebiocalculator.neb.com/#!/)*.*

*- The mass of each fragment can be measured using the NanoDrop instrument, absorbance at 260 nm, or estimated from agarose gel electrophoresis followed by ethidium bromide staining.*

|  |  |  |
| --- | --- | --- |
| **2-3 Fragments Assembly** | **4-6 Fragments Assembly** | **Positive Control \*\*** |
| **Concentration Range of DNA fragments** | 02 - .5 pmols\* | .2 - 1.0 pmols\* | 0 pmols |
| **Total Volume of Fragments (μl)** | 10 |
| **Gibson Assembly Master Mix (2x) (μl)** | 10 | 10 | 10 |
| **Deionized Water (μl)** | #VALUE! | #VALUE! | 0 |
| **Total Volume (μl) \*\*\*** | 20 | 20 | 20 |

*\*Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.*

*\*\* Control reagants are provided for 5 experiments with the Gibson Asembly Kit.*

*\*\*\* If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.*

2. Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled.

*Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases* (for further details see[FAQ section](https://www.neb.com/products/e5510-gibson-assembly-cloning-kit#tabselect1)).

*\*Select the pencil icon and change the timer to 60 minutes if working with 4-6 fragments*

**Timer**: 15 m

3. Store samples on ice or at –20°C for subsequent transformation.
4. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the[chemcial transformation protocol](https://www.neb.com/protocols/2012/12/11/gibson-assembly-transformation-protocol-e5510) or[electro competent cells transformation protocol](https://www.neb.com/protocols/2013/10/29/electrocompetent-cells-transformation-protocol-e5510)

# **Ligation Protocol WITH T4 DNA Ligase (M0202)**

*Note:* T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. *Use*[*NEB calculator*](http://nebiocalculator.neb.com/#!/) *to calculate molar ratios.*

1. Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.
   * *Tip: Aliquot the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.*
2. Set up the following reaction in a microcentrifuge tube on ice:

|  |  |
| --- | --- |
| **Component** | **Volume (μl)** |
| 10X T4 DNA Ligase Buffer | 2 |
| Vector DNA: 50 ng (0.020 pmol) |
| Insert DNA: 37.5 ng (0.060 pmol) |
| Nuclease-free water | 17 |
| T4 DNA Ligase | 1 |
| Total | 20 |

3. Gently mix the reaction by pipetting up and down and microfuge briefly.

4. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.

5. Heat inactivate at 65 degrees C for 10 minutes.

**Timer**: 10 m

6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Use 25 uL DH5α cells, and add 2 uL of reaction mixture.

The free [online CSS cleaner tool](https://html-cleaner.com/css/) helps you to organize style files for websites.

# **1X TAE Buffer**

Formula: M₁ V₁ = M₂ V₂

(50X) V₁ = 1X (1000mL) V₁ = 20mL50X TAE

1. Pour 20mL of 50X TAE into a graduated cylinder, then pour into 1L bottle.
2. Take 1L bottle with 20mL of 50X TAE and fill to 1000mL line (not labelled, indented) with 18mo water.
   * Actually filling bottle with 980 mL of 18mo water. (20 + 980 = 1000mL)
3. Ethidium bromide: (10mg / mL) x (10³ mg / mg) = 1 x 10⁴ μg / mL (concentration in bottle)
   * (1 x 10⁴ μg / mL) V₁ = 0.05 μg / mL (1000 mL) V₁ = 50μL
4. Pipette 50 μL of etBr with 100 μL pipette and release into 1L bottle containing 20 mL of 50X TAE and 980 mL of 18 mo of water
5. Swirl bottle.
6. Cover completely in foil (1L bottle).
7. Label “1X TAE 0.5 μg / mL etBr” on cap with tape.

# **Glycerol Stock**

1. In a 1.5 mL microcentrifuge tube, pipette 100 mL of the 60% glycerol
2. In the same tube, pipette 300 mL of the overnight of your desired plasmid
3. Store tube in a -80 °C freezer

# **LB Agar**

1. Gather material and place them into 1 empty 500 mL Erlenmeyer Flask Make sure to put stir bar in
   * Needed to make into homogenous gel Needs to be sterile
2. Put into Autoclave
   * Lid should not be too tight

# **LB Broth**

1. Place materials in a flask and create a homogenous solution
2. Place flask with material in the autoclave Lid should not be on too tight
3. Lid should be completely tighten after it is finished and taken out of the autoclave

# Overnights

1. Fill your Falcon tube with 3 μL of LB Broth and 3 mL of 25 mg/mL Chloramphenicol
2. Using the toothpick pick a single colony from your plate
3. Place toothpick with colony inside of the Falcon tube
4. Repeat Steps 1-3 as needed
5. Close your Falcon tube(s) and place it/them in the incubator overnight at 37o C

##### Engineering a Cell-Free Hexavalent Chromium Detection System

Email us: iGEM@carrollhs.org

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##### Quick Access

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# Team:US AFRL CarrollHS/Results

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  [Parts Overview](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Parts)
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Results\_Header\_Final

Results\_Header\_Mobile

# Characterization:

# Cell-Free Experiments

Prior to any experimentation, we created 100 µL standard curves using the iGEM Measurement protocols to allow us to convert any data converted our data from arbitrary absorbance and fluorescence units to MEFL/particle.

![](https://static.igem.org/mediawiki/2019/0/08/T--US_AFRL_CarrollHS--ParticleStdCrv.jpg)
![](https://static.igem.org/mediawiki/2019/4/4e/T--US_AFRL_CarrollHS--FluoresceinStdCrv.jpg)

After we created our standard curves, we decided to measure one of our past team’s composite parts from 2017, [PLas\_sfGFP](http://parts.igem.org/Part%3ABBa_K2522001) Part BBa\_K2522001which produces sfGFP in the presence of N-3-oxododecanoyl homoserine lactone (3OC12) to fulfill our Bronze Medal Criterion. Please see our Measurements page for further detail.

Since we were unfamiliar with working with a cell-free system, we wanted to work with optimizing a circuit that we were familiar with: PRhl\_GFPa1.The plasmid was used by our team last year and senses and responds to the quorum-sensing molecule N-butanoyl-L-Homoserine Lactone (C4-HSL). The J23117 native promoter constitutively expresses RhlR, which activates the PRhl promoter site when bound to C4-HSL allowing for the production of the Green-Fluorescent Protein (GFPa1).

Prior to testing it in a cell-free system, we wanted to confirmed its functionality in cells and set up overnights and graphed the results below.

![](https://static.igem.org/mediawiki/2019/e/ea/T--US_AFRL_CarrollHS--PRhl-GFPa1.jpg)

Having confirmed functionality, we moved on to testing it in cell-free.

![](https://static.igem.org/mediawiki/2019/7/7e/T--US_AFRL_CarrollHS--PRhl-GFPa1-CFS.jpg)
![](https://static.igem.org/mediawiki/2019/0/0a/T--US_AFRL_CarrollHS--PRhl-GFPa1-CFS2.jpg)

Since our chromium circuit would contain T7 promoters and native promoters, we tested PRhl-GFPa1 in a T7-optimized kit and looked at the effect of adding bacterial polymerase. Unfortunately, we saw no signal in our PRhl-GFPa1 circuit but did see a signal in our positive control pY71-sfGFP.

We tried to optimize PRhl-GFPa1 in cell-free again but this time we used a native promoter-optimized kit and adding T7 RNA polymerase for our control. We did not see any signal expressed in both the PRhl-GFPa1 plasmid and pY71-sfGFP.

![](https://static.igem.org/mediawiki/2019/1/1e/T--US_AFRL_CarrollHS--PRhl-GFPa1-CFS3.jpg)

# Troubleshooting Cell-Free

We decided to begin troubleshooting our cell-free system and began by testing the functionality of the positive control (pAME215) which has a native promoter and will constitutively express a green fluorescent protein in both the T7-optimized kit and native promoter-optimized kit.

![](https://static.igem.org/mediawiki/2019/2/26/T--US_AFRL_CarrollHS--pAME215-CFS.jpg)

As shown in the graph above, the T7-optimized kit yielded higher results and will work with both types of promoters.

We proceeded to investigate different concentrations of DNA in cell-free and concluded that pAME215 functioned in a T7-optimized kit, however, PRhl-GFPa1 (labeled C4-HSL) did not produce any signal regardless of concentration.

![](https://static.igem.org/mediawiki/2019/2/20/T--US_AFRL_CarrollHS--PRhl-GFPa1-CFS-Tit.jpg)
![](https://static.igem.org/mediawiki/2019/a/af/T--US_AFRL_CarrollHS--PRhl-GFPa1-CFS-Tit2.jpg)

We also wanted to determine whether DMSO, which is what C4-HSL is dissolved in, was potentially affecting our cell-free reaction. Based on the results graphed below, DMSO had no effect on our reactions.

![](https://static.igem.org/mediawiki/2019/0/0e/T--US_AFRL_CarrollHS--DMSO-CFS.jpg)

# pY71-sfGFP

Due to time constraints and since our PRhl-GFPa1 was not functioning in cell-free, we moved on to drying down cell-free reactions of our pY71-sfGFP positive control which we had previously confirmed function in cell-free. We also wanted to determine if adding DNA into the reaction or rehydrating with DNA improved functionality, and if increasing the volume of the reaction increased yield.

![](https://static.igem.org/mediawiki/2019/6/63/T--US_AFRL_CarrollHS--py71-bFMO-Lyoph.jpg)
![](https://static.igem.org/mediawiki/2019/b/bb/T--US_AFRL_CarrollHS--py71-bFMO-Lyoph-25.jpg)

We concluded that adding DNA before in a 25 uL reaction yielded the highest fluorescence.Since we had a success lyophilization, we moved on to applying our circuit on a paper sensor and after one hour of incubation, we saw fluorescence.

![](https://static.igem.org/mediawiki/2019/thumb/3/33/T--US_AFRL_CarrollHS--papersensor.jpg/675px-T--US_AFRL_CarrollHS--papersensor.jpg)

# pY71-bFMO

After receiving the pY71-bFMO plasmid from Lab 2, we confirmed the functionality of it in cells and concluded that with the addition of tryptophan, there was a greater yield of indigo.

![](https://static.igem.org/mediawiki/2019/d/d5/T--US_AFRL_CarrollHS--Indigo.jpg)

We proceeded to test in cell-free but there was no signal detected.

![](https://static.igem.org/mediawiki/2019/9/97/T--US_AFRL_CarrollHS--py71bFMOCFS.jpg)

Since pY71-bFMO was not functional, we took a look at our sequence and discovered that there was an illegal XbaI cut-site in between our T7 promoter and RBS region.

===J18912-sfGFP===
===J18912-bFMO===

The circuit responded to 3OC12 in a dose-dependent manner. We began seeing signal at 10-4 μM and the signal saturated at 10-1 μM. The sensor showed a 60-fold increase in signal upon activation based on the non-linear fit analysis. Analysis was done on GraphPad Prism 5.

We have submitted this characterization in order to fulfill our Bronze Medal Criterion, although we have also used these measurement protocols for the remainder of our experiments. However, our standard curves differ based on factors such as volume and plate type. We’ve graphed all of the other standard curves below.

![](https://static.igem.org/mediawiki/2019/7/7f/T--US_AFRL_CarrollHS--25ul96wellsc.png)
![](https://static.igem.org/mediawiki/2019/2/23/T--US_AFRL_CarrollHS--25ul384wellsc.png)

---

# J18912-sfGFP

We created six overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and either E.coli DH5α cells containing J18912-sfGFP (three overnights) or either E.coli DH5α cells containing pY71-sfGFP(three overnights) to compare the functionality between the two plasmids, and one overnight that only contained 3 mL of LB Broth only to serve as our blank.

![](https://static.igem.org/mediawiki/parts/thumb/a/a6/J18912sfGFPovernights.png/975px-J18912sfGFPovernights.png)

Through these overnights, we were able to confirm the functionality of both plasmids, and we measured the fluorescence and absorbance of each overnight by performing an endpoint read of each overnight in a 96-well flat bottomed plate. In order to obtain data in the range of our standard curve, we diluted the overnights two-fold by mixing 50 µL of each overnight with 50 µL of LB.

We then used the 100 µL standard curve we created using the iGEM Measurement protocols and converted our data from arbitrary absorbance and fluorescence units to MEFL/particle which we graphed below.

![](https://static.igem.org/mediawiki/parts/3/30/J18912-sfGFPcells.png)

**Cell-Free Experiments**

After confirming functionality in cells, we moved on to optimizing our system in cell-free. We set up cell-free reactions and also Although, in cells, there seemed to be no difference in signal of our two plasmids, in cell-free, there was a difference, as shown in the graph below. We believe that this most likely resulted from the difference in DNA concentrations which affects the cell-free system.

![](https://static.igem.org/mediawiki/parts/1/1b/J18912-sfGFPCFS.png)

We also lyophilized our cell-free reactions, and rehydrated them with 25 µL of water. After reading them on the plate reader for four hours, and graphing the data, we were able to confirm functionality of both plasmids, although there was a lower signal in the lyophilization.

![](https://static.igem.org/mediawiki/parts/8/8e/J18912-sfGFPlyozi.png)
![](https://static.igem.org/mediawiki/parts/9/9e/J18912-sfGFPlyozo.png)

**Paper-Based Sensor**

Since both of our plasmids functioned in cell-free, we moved on to applying our plasmids on our paper-based sensor. After one hour of incubation at 30 °C, we were able to confirm functionality and see signal for both plasmids.

![](https://static.igem.org/mediawiki/parts/thumb/0/07/J18912sfGFPpapersensor.png/975px-J18912sfGFPpapersensor.png)

**J18912-bFMO**

We created three overnights that contained 3 mL of LB Broth, 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO and three overnights that contained 2.4 mL of LB Broth, 600 µL of Tryptophan (a precursor to indole), 3 µL of the antibiotic Kanamycin (50 ng/µL) and E.coli DH5α cells containing J18912-bFMO) to compare the functionality between the two plasmids, and one overnight that only contained 3 mL of LB Broth only to serve as our blank.

![](https://static.igem.org/mediawiki/parts/8/84/BFMOpathway.png)

![](https://static.igem.org/mediawiki/parts/thumb/6/6d/J18912bFMOovernights.png/975px-J18912bFMOovernights.png)

To measure indigo production, we performed an extraction protocol on these overnights to extract indigo using DMSO and measured the absorbance on the plate reader. To convert the raw data, we made an indigo standard curve (shown below) and graphed the data below:

![](https://static.igem.org/mediawiki/parts/3/32/Indigostandardcurve.png)

![](https://static.igem.org/mediawiki/parts/thumb/e/e4/J18912bFMO1.png/600px-J18912bFMO1.png)
![](https://static.igem.org/mediawiki/parts/thumb/b/b6/J18912bFMO%2Btrp.png/600px-J18912bFMO%2Btrp.png)

![](https://static.igem.org/mediawiki/parts/1/1c/J18912bFMOovenrights.png)

As shown in the graph above, there was a greater yield of indigo in the overnights that contained additional tryptophan. After we measured the overnights, we moved on to optimizing our system in cell-free. We set up cell-free reactions and graphed the results below.

![](https://static.igem.org/mediawiki/parts/6/6e/J18912-bFMO_graph.png)

Unfortunately, the only “response” we saw in cell-free were reactions with indole, which we believe was due to the indole crashing out of solution. Although we saw indigo production in cells, we did not see similar results in cell-free which will require further optimization.

##### Engineering a Cell-Free Hexavalent Chromium Detection System

Email us: iGEM@carrollhs.org

##### Contact Us

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* [GitHub](https://github.com/us-afrl-carrollhs)

##### Quick Access

* [Meet the Team](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Team)
* [Medal Criteria](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Medal)
* [Project Description](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/Description)
* [Wiki Guide](https://2019.igem.org/Team%3AUS_AFRL_CarrollHS/WikiGuide)
* [Judging Form](https://igem.org/2019_Judging_Form?team=US_AFRL_CarrollHS)

© 2019 Copyright:
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