iGEM 2018 — Team Lambert_GA

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# Team:Lambert GA/Notebook

[![](https://static.igem.org/mediawiki/2018/2/2d/T--Lambert_GA--Captivate.png)](https://2018.igem.org/Team%3ALambert_GA)

[JUDGING FORM](https://igem.org/2018_Judging_Form?team=Lambert_GA)

MODELING

[CALM](https://2018.igem.org/Team%3ALambert_GA/CALM_MODEL)
[ELECTROPEN](https://2018.igem.org/Team%3ALambert_GA/EPEN_MODEL_RESULTS)

CALM

[BACKGROUND](https://2018.igem.org/Team%3ALambert_GA/CALM_BACKGROUND_SOLUTION)
[DATA & METHODS](https://2018.igem.org/Team%3ALambert_GA/CALM_METHODS)
[RESULTS](https://2018.igem.org/Team%3ALambert_GA/CALM_RESULTS)
[DISCUSSION](https://2018.igem.org/Team%3ALambert_GA/CALM_DISCUSSION)
[COLOR Q](https://2018.igem.org/Team%3ALambert_GA/COLOR-Q)
[SUPPLEMENTARY](https://2018.igem.org/Team%3ALambert_GA/CALM_SUPPLEMENTARY)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/CALM_REFERENCES)

QSYSTEM

[COLOR Q](https://2018.igem.org/Team%3ALambert_GA/COLOR-Q)
[CHROME Q](https://2018.igem.org/Team%3ALambert_GA/CHROME-Q)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/MEASUREMENT_REFERENCES)

ELECTROPEN

[PRINCIPLES & DESIGN](https://2018.igem.org/Team%3ALambert_GA/Applied_Design)
[MODEL & THEORY](https://2018.igem.org/Team%3ALambert_GA/EPEN_MODEL_RESULTS)
[PRODUCT DESIGN](https://2018.igem.org/Team%3ALambert_GA/Entrepreneurship)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/ELECTROPEN_REFERENCES)

HUMAN PRACTICES

[INTEGRATED HUMAN PRACTICES](https://2018.igem.org/Team%3ALambert_GA/Human_Practices_Overview)
[EDUCATION & ENGAGEMENT](https://2018.igem.org/Team%3ALambert_GA/Public_Engagement)
[COLLABORATIONS](https://2018.igem.org/Team%3ALambert_GA/Collaborations)

SAFETY

PARTS

[BASIC PARTS](https://2018.igem.org/Team%3ALambert_GA/Basic_Part)
[COMPOSITE PARTS](https://2018.igem.org/Team%3ALambert_GA/Composite_Part)

PROJECT

[DESCRIPTION](https://2018.igem.org/Team%3ALambert_GA/Description)
[BACKGROUND](https://2018.igem.org/Team%3ALambert_GA/Cholera_Background)
[DESIGN](https://2018.igem.org/Team%3ALambert_GA/Design)
[EXPERIMENTS](https://2018.igem.org/Team%3ALambert_GA/Experiments)
[LAB NOTEBOOK](https://2018.igem.org/Team%3ALambert_GA/Notebook)
[INTERLAB](https://2018.igem.org/Team%3ALambert_GA/InterLab)
[RESULTS](https://2018.igem.org/Team%3ALambert_GA/Results)
[DEMONSTRATE](https://2018.igem.org/Team%3ALambert_GA/Demonstrate)
[THE VISION](https://2018.igem.org/Team%3ALambert_GA/Improve)

TEAM

[TEAM MEMBERS](https://2018.igem.org/Team%3ALambert_GA/Team)
[ATTRIBUTIONS](https://2018.igem.org/Team%3ALambert_GA/Attributions)

[

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N O T E B O O K

**[CLONING WORKFLOW](#target1)**

**[NOTEBOOK](#target2)**

**[BIOBRICK NOTEBOOK](#target3)**

**VIEW OUR CLONING WORKFLOW**

Cloning Workflow

Click here to download your own [Ligation Calculator](https://static.igem.org/mediawiki/2017/a/a5/Copy_of_Ligation_Calculator.xlsx).

*Cloning Workflow*

**VIEW OUR TEAM'S NOTEBOOKS**

Team Notebook

*Team Notebook*

**VIEW OUR BIOBRICK NOTEBOOKS**

Biobrick Notebook

*Biobrick Notebook*


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

# Team:Lambert GA/Experiments

[![](https://static.igem.org/mediawiki/2018/2/2d/T--Lambert_GA--Captivate.png)](https://2018.igem.org/Team%3ALambert_GA)

[JUDGING FORM](https://igem.org/2018_Judging_Form?team=Lambert_GA)

MODELING

[CALM](https://2018.igem.org/Team%3ALambert_GA/CALM_MODEL)
[ELECTROPEN](https://2018.igem.org/Team%3ALambert_GA/EPEN_MODEL_RESULTS)

CALM

[BACKGROUND](https://2018.igem.org/Team%3ALambert_GA/CALM_BACKGROUND_SOLUTION)
[DATA & METHODS](https://2018.igem.org/Team%3ALambert_GA/CALM_METHODS)
[RESULTS](https://2018.igem.org/Team%3ALambert_GA/CALM_RESULTS)
[DISCUSSION](https://2018.igem.org/Team%3ALambert_GA/CALM_DISCUSSION)
[COLOR Q](https://2018.igem.org/Team%3ALambert_GA/COLOR-Q)
[SUPPLEMENTARY](https://2018.igem.org/Team%3ALambert_GA/CALM_SUPPLEMENTARY)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/CALM_REFERENCES)

QSYSTEM

[COLOR Q](https://2018.igem.org/Team%3ALambert_GA/COLOR-Q)
[CHROME Q](https://2018.igem.org/Team%3ALambert_GA/CHROME-Q)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/MEASUREMENT_REFERENCES)

ELECTROPEN

[PRINCIPLES & DESIGN](https://2018.igem.org/Team%3ALambert_GA/Applied_Design)
[MODEL & THEORY](https://2018.igem.org/Team%3ALambert_GA/EPEN_MODEL_RESULTS)
[PRODUCT DESIGN](https://2018.igem.org/Team%3ALambert_GA/Entrepreneurship)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/ELECTROPEN_REFERENCES)

HUMAN PRACTICES

[INTEGRATED HUMAN PRACTICES](https://2018.igem.org/Team%3ALambert_GA/Human_Practices_Overview)
[EDUCATION & ENGAGEMENT](https://2018.igem.org/Team%3ALambert_GA/Public_Engagement)
[COLLABORATIONS](https://2018.igem.org/Team%3ALambert_GA/Collaborations)

SAFETY

PARTS

[BASIC PARTS](https://2018.igem.org/Team%3ALambert_GA/Basic_Part)
[COMPOSITE PARTS](https://2018.igem.org/Team%3ALambert_GA/Composite_Part)

PROJECT

[DESCRIPTION](https://2018.igem.org/Team%3ALambert_GA/Description)
[BACKGROUND](https://2018.igem.org/Team%3ALambert_GA/Cholera_Background)
[DESIGN](https://2018.igem.org/Team%3ALambert_GA/Design)
[EXPERIMENTS](https://2018.igem.org/Team%3ALambert_GA/Experiments)
[LAB NOTEBOOK](https://2018.igem.org/Team%3ALambert_GA/Notebook)
[INTERLAB](https://2018.igem.org/Team%3ALambert_GA/InterLab)
[RESULTS](https://2018.igem.org/Team%3ALambert_GA/Results)
[DEMONSTRATE](https://2018.igem.org/Team%3ALambert_GA/Demonstrate)
[THE VISION](https://2018.igem.org/Team%3ALambert_GA/Improve)

TEAM

[TEAM MEMBERS](https://2018.igem.org/Team%3ALambert_GA/Team)
[ATTRIBUTIONS](https://2018.igem.org/Team%3ALambert_GA/Attributions)

[

Your browser does not support video.
](https://static.igem.org/mediawiki/2018/c/cc/T--Lambert_GA--Experiments.mp4)

E X P E R I M E N T S

**[WET LAB SUMMARY](#target1)**

**[WORKFLOW](#target2)**

**[MATERIALS](#target3)**

**[WATER COLLECTION AND ANALYSIS](#target4)**

**[REFERENCES](#target5)**

**Wet Lab Summary**

Lambert iGEM utilized a toehold switch and a trigger - which corresponds with RNA sequences of the ctxB1 cholera gene - in a biosensor system. If the trigger sequence is present and binds to the toehold switch, then a blue pigment produced by LacZ appears in the cells.

![](https://static.igem.org/mediawiki/2018/0/07/T--Lambert_GA--Trigger_Toehold.png)

*The construct above displays the proof of concept T7 LacZ switch and trigger. The switch consists of T7 promoter, a strong constitutive promoter, along with LacZ as the reporter gene. The trigger was cloned into a high copy plasmid while the switch was cloned into a low copy plasmid to ensure the replication of two different plasmids for the E. coli cell to produce proportional amounts.*

**Work Flow**

The 2018 Lambert iGEM team conducted the following experiments to assemble the proof the concept T7 LacZ toehold switch and Cholera switch.
We ordered three potential Cholera toehold switches and triggers and proceeded to perform PCRs on each of the parts. All switches and triggers were amplified and validated through gel electrophoresis [Figure 1.]. With purified PCR products of the switches, triggers, and their correlating vectors, we ligated each of these parts into their vectors through Gibson Assembly. Dual plasmid transformations and single transformations were conducted with BL21 T7 E. coli

Click on the step to be directed to the procedure!
**1. [Miniprep](#Step1)
2. [Nanodrop](#Step2)
3. [Digest](#Step3)
4. [Gel](#Step4)
5. [Ligation](#Step5)
6. [Transformation/electroporation, Plate](#Step6)
7. [Colony PCR (Screening)](#Step7)
8. [Gel](#Step4)
9. [Inoculate correct colony to a liquid culture](#Step8)
10. [Miniprep](#Step1)
11. [Nanodrop](#Step2)
12. Sequence**

**Materials**

**Miniprep:** grown culture, microcentrifuge, 2 1.5mL microcentrifuge tubes, mini column and collection tube, Solution I, Solution II, Solution III, HBC Wash Buffer, DNA Wash Buffer, Elution Buffer, micropipette and tips

**Nanodrop:** nanodrop machine, miniprepped DNA, Kimtech wipes, micropipette and tips

**Digest:** miniprepped DNA, dH₂O, 10X RE-Mix, standard restriction enzyme, micropipettes and tips

**Gel:** agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA

**Ligation:** vector, parts 1 and 2, ligase buffer, ligase, Antarctic phosphatase, microcentrifuge tube, ice, micropipette and tips

**Transformation:** ice, ligation mixture, competent cells, incubator, LB media, microcentrifuge tubes, micropipette and tips Plate: agar plate, micropipette and tips, beads

**Colony PCR:** dH₂O, buffer, VF₂, VR, Q5 polymerase, dNTP, DNA dilution, micropipette and tips, PCR tubes, thermocycler, ice

**Gel:** agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA

**Inoculate:** LB media, dilution, micropipette and tips

**Miniprep (using Omega Protocol)**

1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube.

1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media. (Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.)

* 1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.

1.3 Add 250uL of Solution I mixed with RNase A (pre-added). Vortex to mix thoroughly. Transfer the suspension into a new 1.5mL microcentrifuge tube.

1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate.

* 1.4.1 Once Solution II is added, do not let it sit for more than 5 minutes!

1.5 Add 350uL of Solution III. Invert several times until white precipitate forms. Centrifuge at 13,000xg or 17,900rcf for 10 minutes. A compact white pellet should form at the bottom of the tube.

1.6 Insert a mini-column into a 2mL collection tube.

1.7 Transfer the clear supernatant into the mini-column using a micropipette. Centrifuge at the maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.

* 1.7.1 Be careful not to get any parts of the pellet! Tilt at an angle with the pellet at the top when micropipetting is advisable.

* 1.7.2 Think about what you are discarding versus what you want to keep!

1.8 Add 500uL of the HBC Wash Buffer diluted in isopropanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.

* 1.8.1 All wash buffers will be centrifuged for 1 minute.

1.9 Add 700uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.

1.10 Centrifuge the empty mini column at the maximum speed (13,000xg) for 2 minutes to remove the ethanol.

1.11 Transfer the mini-column to a nuclease-free 1.5mL microcentrifuge tube.

1.12 Add 50uL of Elution Buffer (or sterile deionized water). Let it sit in room temperature for 60 seconds. Centrifuge at maximum speed (13,000xg) for 60 seconds.

1.13 Store eluted DNA at -20℃.

**Nanodrop**

2.1 Vortex before nanodrop.

2.2 Wipe down the nanodrop machine with Kimtech wipes to make it sterile.

2.3 Set the program to analyze nucleic acids [because you are dealing with plasmid DNA].

2.4 Do a blank test to ensure that the platform is sterile.

2.5 Load 1uL of the miniprepped DNA onto the platform.

2.5.1 (Have steady hands. The sample needs to be in the center for best results.)

2.6 Click “measure” on the nanodrop for analysis.

2.7 Write down measurements for the concentration of DNA (in ng/uL), A260, A280, 260/280 (should be around 1.8), and 260/230 (should be around 2.1).

**Digest**

3.1 Dilute up to 1ug DNA to 17uL with dH₂O.

* 3.1.1 Take concentration of DNA from nanodrop and convert from ng/uL to ug/uL. Next, set up a proportion to find out how many uL you need to get 1 ug of DNA.
* 3.1.2 20uL (total reaction) - 2uL RE-Mix - 1uL standard enzyme = uL dH₂O

3.2 Use a microcentrifuge tube to put the reaction in. Put in the contents in this order: water, DNA, enzymes.

* 3.2.1 Add 2uL of the 10X RE-Mix and 1uL of the standard enzyme.

+ 3.2.1.1 E and X = 10X RE-Mix
+ 3.2.1.2 S and P = standard enzymes

3.3 Incubate at 37℃ for 1 hour for standard enzymes, then at 80℃ for deactivation.

**Gel**

4.1 Set up the chamber and put in the gel. Make sure the wells of the gel is at the end of the chamber so that the DNA runs to red.

4.2 Pour the TAE buffer evenly to completely cover the gel.

4.3 Using a micropipette, put 3uL of DNA in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more DNA to be put in each well.

4.4 Connect the electrodes by closing the box and connecting them to the power supply. Make sure the power supply is set for 120 volts and 60 minutes.

4.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber.

**Ligation**

5.1 Use Antarctic phosphatase on the backbone to increase the likelihood of part insertion and decrease backbone closure.Make calculations using a 3:1 molar ratio of insert to backbone. Refer to the two tables below.

5.2 Put in each component in a microcentrifuge tube while on ice. They should be pipetted into the tube in this order: water, DNA, ligase buffer, ligase.

5.3 The ligase buffer should be thawed and resuspended at room temperature.

* 5.3.1 Gently mix by pipetting up and down and microfuge briefly.

5.4 Incubate at room temperature for 1 hour at 37℃

**Transformation**

6.1 Thaw materials on ice for 5 minutes.

6.2 Put 10uL of ligation mixture into 100uL competent cells in a microcentrifuge tube.

6.3 Flick the tube to mix.
6.5 Add 200uL of LB media.

6.6 Incubate at 37℃ for one hour.

6.7 Plate 150uL of cells onto a plate. Make sure plate has the correct antibiotic (based on vector backbone)! Grow overnight.

**Electroporation**

(for 400 ml culture, adjust as appropriate for smaller volumes)

**Preparing Electrocompetent cells:**

6.1 Grow overnight 5 ml culture

6.2 Dilute 1:100 in fresh media

\*\* preparation: chill big centrifuge to 4C, chill autoclaved water and 10% glycerol soln

6.3 Grow to OD of about 0.6 (isolating in exponential phase most important)

6.4 Pour cells into 50 ml conical tubes, on ice

6.5 Keep on ice for 10 min

6.6 Centrifuge cells (all spins done at 2500 xg, 6 min), discard supernatant

6.7 Add 13 ml of ice cold sterile water to each tube, resuspend by pipetting up and down

6.8 Combine into two total tubes.

6.9 Centrifuge, repeat ice cold water resuspension

6.10 Do same thing twice with 25 ml of ice cold 10% glycerol

6.11 Resuspend in 4 ml (concentrating 100x from initial culture) of ice cold 10% glycerol

6.12 Aliquot into microcentrifuge tubes

6.13 Store in -80 freezer

**Electroporation:**

6.14 Take 50 ul of electrocompetent cells, and 10-100 ng of PCR product (don’t add more than 2ul) for 6.15 knockouts, 0.1-10 ng of plasmid

6.16 Flick to mix

6.17 Transfer to chilled electroporation cuvette

6.18 Electroporate (machine in Anton’s lab downstairs)

* 6.18.1 (first setting), target time constant: greater than or equal to 5 ms
* 6.19 Add 1ml of prewarmed LB to cuvette

6.20 Transfer cell/LB mixture to microcentrifuge tube

6.21 Recover with shaking in incubator for 1.5 hr

**Colony PCR**

7.1 Pick colonies with a combination of phenotypes i.e. large/small, red/white. Dilute each colony in 40uL dH₂O, 1uL DNA from ligation if transformation is successful.

* 7.1.1 If necessary, do a quick spin to make sure all the liquid is at the bottom.

7.2 Make the following master mix on ice in this order: 63uL dH₂O, 20uL buffer, 5uL VF₂ primer, 5uL VR primer, 2uL dNTP, 1uL Q5 polymerase.

7.3 Aliquot the master mixes into PCR tubes, then add 1uL of the DNA dilution.

* 7.3.1 Make sure PCR tubes are labeled properly and carefully!

7.4 Transfer the PCR tubes to a PCR machine and begin thermocycling.

* 7.4.1 Initial Denaturation: 98℃ for 30 seconds
* 7.4.2 25-35 Cycles: 98℃ for 5-10 seconds, 50-72℃ for 10-30 seconds, 72℃ for 20-30 seconds/kb
* 7.4.3 Final Extension: 72℃ for 2 minutes
* 7.4.4 Hold 4-10℃

**Gel**

8.1 Set up the chamber and put in the gel. Make sure the wells of the gel is at the end of the chamber so that the DNA runs to red.

8.2 Pour the TAE buffer evenly to completely cover the gel.

8.3 Using a micropipette, put 3uL of DNA in each well and 6uL for the ladder [if using a thin gel]. Thicker gels will require more DNA to be put in each well.

8.4 Connect the electrodes by closing the box and connecting them to the power supply. Make sure the power supply is set for 120 volts and 60 minutes.

8.5 Turn on the power supply and make sure bubbles are rising on the sides of the chamber.

**Inoculate Liquid Culture**

9.1 Get the remaining 39uL of colony dilution.

9.2 Get LB media and make sure to use the appropriate antibiotic resistance.

9.3 Mix the colony dilution into the media.

9.4 Grow overnight.

**Water Collection and Analysis**

**Water Collection**

**Prepping the Bottle**

1.1 Obtain 4 nalgene bottles (22.75 cm in length, 5.5 cm in width).

1.2 Drill a 1.9 cm (preferably 2 cm) hole in the bottom of the bottle with a ¾ in drill bit.

1.3 Run the bottle through tap water to get any plastic bits out of the bottle. Spray the interior of the bottle with isopropyl alcohol. Rinse with soap and water to remove any alcohol.

1.4 Allow the bottles to dry by placing them on a drying rack.

**Prepping Muslin Cloth**

1. Obtain 90 cm by 90 cm muslin cloth.
2. Cut the muslin cloth into 6 cm. by 6 cm pieces.
3. Rinse with soap under flowing tap water.
4. Allow the muslin cloth to dry.
5. Fold in half.
6. Place the muslin cloth into an ice bottle.
7. Fill the bottle ¾th of the way with the cloth

**Prepping Cheese Cloth**

1. Obtain a 5.5 meter by 19.4 cm.
2. Cut the cheese cloth into 20.5 cm by 15 cm.
3. Rinse the cheesecloth in running tap water and soap.
4. Create 3-4 folds and pack the ice bottle.
5. Use a long rod to help push the cloth into the bottle and make the bottle compact.
6. Fill the bottle ¾th of the way with the cloth.

**Prepping T-shirt Cloth**

1. Obtain 1 t-shirt
2. Cut into 5 cm by 11 cm strips
3. Fold strips 2 times and pack ice bottle
4. Fill bottle ½ way with the cloth

**Prepping Coffee Cloth**

1. Obtain a pack of large coffee filter
2. Microwave filters to sterilize
3. Cut filters in ½
4. Fold pieces 1-2 times and pack ice bottle
5. Fill bottle ¾th of the way with the filters

**Prepping Carb Cloth**

1. Add 500 ml of dH20 and 17.5g of agar to a stock jar
2. Autoclave solution
3. Add 500µl of carb
4. Pour plates as usual

**Creating Liquid Culture**

1. Add 5ml of LB to a 15ml culture tube
2. Inoculate a few colonies of cells

**Obtaining Water**

1. Obtain 1000 ml of creek water from any nearby water body by placing a stock jar within the water and allowing the water to flow into the stock jar.

![](https://static.igem.org/mediawiki/2018/3/38/T--Lambert_GA--WaterCollection.jpg)

**Filtration**

1. Run distilled water through all the filters until all filters are wet.
2. Get five 600 ml beakers
3. Combine the 5 ml liquid culture of E.coli to the 1000ml creek water. Stir the stock jar until the liquid culture is thoroughly mixed with the creek water.
4. Measure 100ml of the creek water spiked with E. Coli.
5. Run the 100 ml of creek water through the muslin filter bottle. Make sure a labeled 600 ml beaker is placed under the bottle to catch filtered water.
6. Repeat steps 4 and 5 with the negative control (no filter), cheesecloth, t-shirt, coffee filters.
7. Run the creek water spiked with E. coli through the Micron Filter (Positive Control)

1. Pour 100 ml of the creek water spike with E. Coli to the top cup of the micron filter.
2. Attach the filtration vacuum to the side of the micron filter. Turn on the vacuum so the water is filtered through the .2 micron filter.
3. Allow the water to be filtered through the micron filter and stop the vacuum when all the water has filtered to the bottom container of the micron filter.

**Serial Dilution and Plating**

1. Obtain 6 centrifuge tubes.
2. Plate 150 microliters of each of the filtered solutions in the 6 600ml beakers into corresponding carb plates. Use glass beads to spread the cultures throughout the carb plates.
3. Fill a beaker with 10 mL of distilled water.
4. Pipette 15 microliters of the filtered water that ran through the muslin and dispense in a centrifuge tube. Label the centrifuge tube, muslin.
5. Pipette 185 microliters of distilled water and dispense in the same centrifuge tube.
6. Repeat steps 12 and 13 with the negative control (no filter), cheesecloth, t-shirt, coffee filters, and the micron (positive) filter.
7. Plate 150 microliters of the solutions from the microcentrifuge to the corresponding carb plates. Use glass beads to spread the cultures throughout the carb plates.
8. Place the plates in the incubator and incubate at 37 degrees Celsius for 24 hours.
9. After 24 hours observe the results and note use colony forming units.

![](https://static.igem.org/mediawiki/2018/thumb/a/a0/T--Lambert_GA--experiments2.jpeg/450px-T--Lambert_GA--experiments2.jpeg
)
![](https://static.igem.org/mediawiki/2018/thumb/7/75/T--Lambert_GA--experiments3.png/800px-T--Lambert_GA--experiments3.png.jpeg)

*Picture of serial dilution and growth on plate (Image on the left) and various cloths used for testing the number of colony forming units (Image on the right).*

**Data**

| - | Coffee Filter | T-shirt | Cheese Cloth | Muslin | + |
| --- | --- | --- | --- | --- | --- |
| 37 | 89 | Too many to count/multiple types | Multiple types of bacteria | Too many to count | 0 |
| 5 | 1 | Too many to count/multiple types | Multiple types of bacteria | Too many to count | 0 |

**Analysis**

The negative control showed a limited growth of e.coli colonies. The positive control had no growth meaning the microfilter was effectively filtered out all growth. Having used a blue t-shirt, we found that the water ran through turned blue. For the cheesecloth, only 50ml of water came out and it appeared to be yellow. All tested variables had more growth than the negative control, showing that there was a source of contamination in the experiment. However, coffee filter and muslin appeared to have the least e.coli growth showing they could possibly be effective for filtration. This experiment did not conclusively show whether coffee filter or muslin was more effective so we will repeat the experiment with coffee filters and muslin.

![](https://static.igem.org/mediawiki/2018/3/36/T--Lambert_GA--experimentstable.png)
![](https://static.igem.org/mediawiki/2018/2/2a/T--Lambert_GA--experiments1.png)

**References**

[1] *EXAMINATION OF FOOD AND ENVIRONMENTAL SAMPLES*[PDF]. (n.d.). Centers for Disease Control.

[2] London School of Hygiene & Tropical Medicine. (2018, May 03). Upcoming rainy season likely to trigger renewed cholera outbreak in Yemen. Retrieved from https://www.lshtm.ac.uk/newsevents/news/2018/upcoming-rainy-season-likely-trigger-renewed-cholera-outbreak-yemen

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

# Team:Lambert GA/Results

[![](https://static.igem.org/mediawiki/2018/2/2d/T--Lambert_GA--Captivate.png)](https://2018.igem.org/Team%3ALambert_GA)

[JUDGING FORM](https://igem.org/2018_Judging_Form?team=Lambert_GA)

MODELING

[CALM](https://2018.igem.org/Team%3ALambert_GA/CALM_MODEL)
[ELECTROPEN](https://2018.igem.org/Team%3ALambert_GA/EPEN_MODEL_RESULTS)

CALM

[BACKGROUND](https://2018.igem.org/Team%3ALambert_GA/CALM_BACKGROUND_SOLUTION)
[DATA & METHODS](https://2018.igem.org/Team%3ALambert_GA/CALM_METHODS)
[RESULTS](https://2018.igem.org/Team%3ALambert_GA/CALM_RESULTS)
[DISCUSSION](https://2018.igem.org/Team%3ALambert_GA/CALM_DISCUSSION)
[COLOR Q](https://2018.igem.org/Team%3ALambert_GA/COLOR-Q)
[SUPPLEMENTARY](https://2018.igem.org/Team%3ALambert_GA/CALM_SUPPLEMENTARY)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/CALM_REFERENCES)

QSYSTEM

[COLOR Q](https://2018.igem.org/Team%3ALambert_GA/COLOR-Q)
[CHROME Q](https://2018.igem.org/Team%3ALambert_GA/CHROME-Q)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/MEASUREMENT_REFERENCES)

ELECTROPEN

[PRINCIPLES & DESIGN](https://2018.igem.org/Team%3ALambert_GA/Applied_Design)
[MODEL & THEORY](https://2018.igem.org/Team%3ALambert_GA/EPEN_MODEL_RESULTS)
[PRODUCT DESIGN](https://2018.igem.org/Team%3ALambert_GA/Entrepreneurship)
[REFERENCES](https://2018.igem.org/Team%3ALambert_GA/ELECTROPEN_REFERENCES)

HUMAN PRACTICES

[INTEGRATED HUMAN PRACTICES](https://2018.igem.org/Team%3ALambert_GA/Human_Practices_Overview)
[EDUCATION & ENGAGEMENT](https://2018.igem.org/Team%3ALambert_GA/Public_Engagement)
[COLLABORATIONS](https://2018.igem.org/Team%3ALambert_GA/Collaborations)

SAFETY

PARTS

[BASIC PARTS](https://2018.igem.org/Team%3ALambert_GA/Basic_Part)
[COMPOSITE PARTS](https://2018.igem.org/Team%3ALambert_GA/Composite_Part)

PROJECT

[DESCRIPTION](https://2018.igem.org/Team%3ALambert_GA/Description)
[BACKGROUND](https://2018.igem.org/Team%3ALambert_GA/Cholera_Background)
[DESIGN](https://2018.igem.org/Team%3ALambert_GA/Design)
[EXPERIMENTS](https://2018.igem.org/Team%3ALambert_GA/Experiments)
[LAB NOTEBOOK](https://2018.igem.org/Team%3ALambert_GA/Notebook)
[INTERLAB](https://2018.igem.org/Team%3ALambert_GA/InterLab)
[RESULTS](https://2018.igem.org/Team%3ALambert_GA/Results)
[DEMONSTRATE](https://2018.igem.org/Team%3ALambert_GA/Demonstrate)
[THE VISION](https://2018.igem.org/Team%3ALambert_GA/Improve)

TEAM

[TEAM MEMBERS](https://2018.igem.org/Team%3ALambert_GA/Team)
[ATTRIBUTIONS](https://2018.igem.org/Team%3ALambert_GA/Attributions)

[

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R E S U L T S

**[PROOF OF CONCEPT RESULTS](#target1)**

**[CHOLERA TOEHOLD SWITCH](#target2)**

**Proof of Concept Results**

In order to utilize LacZ color expression as a biosensor mechanism, Lambert iGEM obtained a LacZ toehold construct assembled with a T7 promoter from the Styczynski Lab at the Georgia Institute of Technology. When assembled with a distinct RNA sequence that is complementary to the trigger sequence, the LacZ operon is induced and subsequently breaks down Xgal and produces galactose and a blue pigment. This color can then be characterized by the shade of the blue pigment expression. This part is transformed in pSB3C5 instead of pSB1C3 because when LacZ is induced in a high copy plasmid such as pSB1C3, it drains the metabolism of a cell.

![](https://static.igem.org/mediawiki/2018/0/07/T--Lambert_GA--Trigger_Toehold.png)

*The construct above displays the proof of concept T7 LacZ switch and trigger. The switch consists of T7 promoter, a strong constitutive promoter, along with LacZ as the reporter gene. The trigger was cloned into a high copy plasmid while the switch was cloned into a low copy plasmid to ensure the replication of two different plasmids for the E. coli cell to produce proportional amounts.*

Lambert iGEM used sequences and DNA shared from the Styczynski Lab at the Georgia Institute of Technology to build a T7, Toehold, LacZ switch to be applied as a biosensor. We tested the Biobricked T7 Toehold LacZ Switch BBa\_K2550000, the Biobricked T7 Trigger Sequence BBa\_K2550001 T7 Toehold LacZ Switch obtained from GATech, and the pSB6A1 T7 Trigger on Luria broth plates with chloramphenicol, carbenicillin, and Xgal antibiotic resistance.

![](https://static.igem.org/mediawiki/2018/3/38/T--Lambert_GA--results4.jpeg)

*Figure 1: (Top left) Biobricked T7 Toehold LacZ Switch BBa\_K2550000 transformed on chloramphenicol, carbenicillin, and Xgal. (Top right) Biobricked Trigger Sequence BBa\_K2550001 transformed on chloramphenicol antibiotic resistance. (Bottom left) pSB6A1 T7 Toehold LacZ Switch transformed on chloramphenicol, carbenicillin, and Xgal. (Bottom right) Dual plasmid with biobricked toehold sequence and trigger sequence.*

The dual plasmid transformation using the pSB6A1 trigger and biobricked toehold was successful. Since the blue expression was only evident on the dual plasmid plate and not on the plate that contained the toehold, the trigger is successful in inducing LacZ gene expression.

The transformations of the biobricked toehold construct were unsuccessful because they produced a blue color. The blue might be a result of the orthogonal switch not binding as expected or a result of overexpression from a too strong promoter. Since the trigger sequence was not transformed with the construct, the colonies should appear white as the toehold only reveals the RBS and starting sequence of LacZ when the trigger sequence. This could be due to a leaky expression of LacZ under the strong T7 promoter. The dual plasmid transformation confirmed that neither the biobricked trigger nor the biobricked toehold sequence worked in conjunction with each other although they worked in conjunction with similar parts not in pSB1C3.

![](https://static.igem.org/mediawiki/parts/a/a4/T--Lambert_GA--JSPICS.png)

*Figure 2: (Right) Dual Plasmid Transformation of T7 Trigger Sequence and T7 Toehold LacZ on chloramphenicol, carbenicillin, and Xgal antibiotic resistance. (Left) T7 Toehold LacZ transformation on chloramphenicol antibiotic resistance. These show the expected results of our constructs.*

![](https://static.igem.org/mediawiki/2018/a/a7/T--Lambert_GA--sequencing1.png)

*Figure 3: Sequencing results of the trigger sequence that confirmed the part was correct.*

We aligned the sequencing results to the original trigger sequence obtained from the second entry in the 144 first generation orthogonal toehold switches collection from the 2017 Green etal paper. Since the sequences matched after alignment, this confirmed that the trigger sequence was correctly cloned.

**Cholera Toehold Switch**

To test the functionality of our Cholera toehold switches, we first performed dual plasmid transformations by electroporating 10 ng/ul of each switch and trigger plasmid into BL21 E. coli. In addition, we performed single transformations on each of the switches to test the on-off functions of our toehold switches. All three of our Cholera switch transformations presented blue colonies. We have hypothesized that this is due to the leakiness of the switches in response to the strength of the T7 promoter causing the blue expression of LacZ when in the presence of Xgal. In addition, the dual plasmid transformations were unsuccessful. This could be due to the low time constants that were produced during electroporation causing the BL21 cells to not have their pores open for enough time preventing efficient uptake of the plasmids. Due to these inconclusive results, Lambert iGEM performed a chemical transformation with these toehold switches.
![](https://static.igem.org/mediawiki/2018/0/0a/T--Lambert_GA--results5.1.jpeg)

*Figure 4: Unsuccessful Electroporation- Plates on top are the single switch transformations. Plates on the bottom are the dual plasmid transformations.*

![](https://static.igem.org/mediawiki/2018/2/25/T--Lambert_GA--results6.jpeg)

*Figure 5: Chemical Transformation- Plates on the top are single switch transformations of the Cholera switches. The plates on the bottom are the dual plasmid transformations of Cholera 3, 2, and 1.*

**Unsuccessful: Leakiness**

Due to the inconclusive results from the electroporation, Lambert iGEM performed a chemical transformation with our Cholera toehold switches and triggers in BL21 E. coli. For the dual plasmid transformation, 50 ng/ul of both switch and trigger plasmids were used with a total of 100 ng of plasmid. Both Cholera 1 and 2 single switch transformations show a strong blue expression demonstrating the same leakiness presented in the electroporation results. However, the Cholera 3 switch was grown on a chloramphenicol plate in the absence of Xgal causing these cells to not express the blue pigment from LacZ. In addition, unlike the dual plasmid results from the electroporation, the chemically transformed cells grew and expressed blue pigment at a lower efficiency than the single switch transformations.

**Sequencing: unsuccessful**

The sequencing results for the Cholera switches show that the Cholera 1 and 2 switches contain the toehold switch from the 2017 Collins paper titled “ Toehold Switches: De-Novo-Designed Regulators of Gene Expression”. Therefore, our Cholera switches did not contain the correct sequences.

![](https://static.igem.org/mediawiki/2018/6/64/T--Lambert_GA--sequencing2.png)

![](https://static.igem.org/mediawiki/2018/2/2f/T--Lambert_GA--sequencing3.png)

![](https://static.igem.org/mediawiki/2018/f/f1/T--Lambert_GA--sequencing4.png)