iGEM 2021 — Team WLC-Milwaukee

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

# Team:WLC-Milwaukee/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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

# Team:WLC-Milwaukee/Engineering

Header

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2021/9/93/T--WLC-Milwaukee--biglogotrnsparent.png "WLC-Milwaukee iGEM Home")](/Team%3AWLC-Milwaukee)

[PROJECT](%5CTeam%3AWLC-Milwaukee%5CDescription)

[Description](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Engineering](%5CTeam%3AWLC-Milwaukee%5CEngineering)
[Design](%5CTeam%3AWLC-Milwaukee%5CDesign)
[Results](%5CTeam%3AWLC-Milwaukee%5CResults)
[Implementation](%5CTeam%3AWLC-Milwaukee%5CImplementation)
[Lab Manual](%5CTeam%3AWLC-Milwaukee%5CCommunication)
[Demonstrate](%5CTeam%3AWLC-Milwaukee%5CProof_Of_Concept)
[Contribution](%5CTeam%3AWLC-Milwaukee%5CContribution)
[Improve](%5CTeam%3AWLC-Milwaukee%5CImprove)

[PARTS](%5CTeam%3AWLC-Milwaukee%5CParts)

[Parts](%5CTeam%3AWLC-Milwaukee%5CParts)
[Part Collection](%5CTeam%3AWLC-Milwaukee%5CPart_Collection)
[Experiments](%5CTeam%3AWLC-Milwaukee%5CExperiments)

[HUMAN PRACTICES](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)

[Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Integrated Human Practices](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices%5CiHP)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)
[Education](%5CTeam%3AWLC-Milwaukee%5CEducation)

[TEAM](%5CTeam%3AWLC-Milwaukee%5CTeam)

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)
[Medals](%5CTeam%3AWLC-Milwaukee%5CMedals)
[Awards](%5CTeam%3AWLC-Milwaukee%5CAwards)

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2021/9/93/T--WLC-Milwaukee--biglogotrnsparent.png "WLC-Milwaukee")WLC-Milwaukee](/Team%3AWLC-Milwaukee)

PROJECT

[Description](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Engineering](%5CTeam%3AWLC-Milwaukee%5CEngineering)
[Design](%5CTeam%3AWLC-Milwaukee%5CDesign)
[Results](%5CTeam%3AWLC-Milwaukee%5CResults)
[Implementation](%5CTeam%3AWLC-Milwaukee%5CImplementation)
[Lab Manual](%5CTeam%3AWLC-Milwaukee%5CCommunication)
[Demonstrate](%5CTeam%3AWLC-Milwaukee%5CProof_Of_Concept)
[Contribution](%5CTeam%3AWLC-Milwaukee%5CContribution)
[Improve](%5CTeam%3AWLC-Milwaukee%5CImprove)

DOCUMENTATION

[Parts](%5CTeam%3AWLC-Milwaukee%5CParts)
[Part Collection](%5CTeam%3AWLC-Milwaukee%5CPart_Collection)
[Experiments](%5CTeam%3AWLC-Milwaukee%5CExperiments)

HUMAN PRACTICES

[Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Integrated Human Practices](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices%5CiHP)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)
[Education](%5CTeam%3AWLC-Milwaukee%5CEducation)

TEAM

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)
[Medals](%5CTeam%3AWLC-Milwaukee%5CMedals)
[Awards](%5CTeam%3AWLC-Milwaukee%5CAwards)

[![Instagram](https://static.igem.org/mediawiki/2021/c/c6/T--WLC-Milwaukee--instagram.png)](https://www.instagram.com/wlcigem/)
![Email](https://static.igem.org/mediawiki/2021/8/8f/T--WLC-Milwaukee--email.iconw.png)

/\*Make text red and bold\*/
.red\_text {
color: #f42434;
font-weight:bold;
}

WLC-Milwaukee 2021 | Engineering

Engineering

![Think](https://static.igem.org/mediawiki/2021/9/9f/T--WLC-Milwaukee--think.png)

The environment-dependent expression of genes—gene regulation—is a notoriously difficult concept. Participation in iGEM requires an intricate understanding of gene regulation, but there are few resources available for those trying to learn. The goal of our project is to create a hands-on laboratory kit to teach concepts that form the foundation of synthetic biology. We concluded that a color change would be best to determine whether gene expression was occurring.

![Plan](https://static.igem.org/mediawiki/2021/0/08/T--WLC-Milwaukee--plan.png)

First, we had to identify a gene that when expressed induced a color change in the presence of its substrate. We then had to place that gene under the control of regulatory elements such that it was only expressed in certain environments. Then, if the environmental conditions were right and the substrate was present the color of the solution would change!

![Design](https://static.igem.org/mediawiki/2021/d/d3/T--WLC-Milwaukee--designeng.png)

The lac operon includes a gene that when expressed induces a color change. The product of lacZ turns blue in the presence of the substrate x-gal. Additionally, the regulatory components of the lac operon are well documented and understood. Their function is dependent on the concentration of glucose and lactose in the environment. Lactose enters the cell through membrane proteins expressed by lacY. Therefore, we decided to insert the lac operon into a plasmid and transform it into *E. coli*.

![Lac Operon](https://static.igem.org/mediawiki/2021/4/46/T--WLC-Milwaukee--niceoperon.png)

![Build](https://static.igem.org/mediawiki/2021/2/24/T--WLC-Milwaukee--build.png)

The regulatory components of the lac operon and lacY were successfully inserted into the plasmid and verified with sequencing. Unfortunately, we did not successfully insert lacZ into the plasmid with the regulatory components and lacY.

![Review](https://static.igem.org/mediawiki/2021/a/af/T--WLC-Milwaukee--review.png)

We concluded that lacZ could not be inserted into the plasmid with the regulatory components and lacY because it would yield a DNA fragment that was too large. Without lacZ we could not achieve the color change we wanted. We needed a different gene!

![Improve](https://static.igem.org/mediawiki/2021/3/3c/T--WLC-Milwaukee--improveeng.png)

We identified two prospective genes: bla and icm. Bla expresses b-lactamase which turns nitrocefin red. Icm expresses the indole-3-carboxylate monooxygenase that turns indole-3-carboxylic acid blue. We decided to insert each gene into its own plasmid and transform it into *E. coli*.

![Lacz to BLA/ICM](https://static.igem.org/mediawiki/2021/8/8e/T--WLC-Milwaukee--lacblaicm.png)

![Rebuild](https://static.igem.org/mediawiki/2021/3/34/T--WLC-Milwaukee--rebuild.png)

We acquired plasmids with the regulatory components and bla/icm genes and transformed them into *E. coli*.

![Blawhole](https://static.igem.org/mediawiki/2021/f/fd/T--WLC-Milwaukee--blawhole.png)

![Test](https://static.igem.org/mediawiki/2021/1/1d/T--WLC-Milwaukee--test.png)

We tested out bla constructs and noticed a very obvious color change. Even more, we observed color changes consistent with the expected regulation! Success!

![Color Change](https://static.igem.org/mediawiki/2021/8/87/T--WLC-Milwaukee--colorchange.png)

![Review](https://static.igem.org/mediawiki/2021/a/af/T--WLC-Milwaukee--review.png)

Although we were successful in obtaining a construct that changed colors, our project was not completed. To accurately teach all aspects of gene regulation, an understanding of the specific regulatory regions is imperative. We sought to inactivate certain promoters, repressors, and activators in our construct to accomplish this.

![Rebuild](https://static.igem.org/mediawiki/2021/3/34/T--WLC-Milwaukee--rebuild.png)

Specifically, we used site-directed mutagenesis to inactivate the lacI promoter, lacP, the CAP binding site, the lacI open reading frame, lacO, and the ribosome binding site for bla. We sent the mutants for sequencing and found that the lacI promoter, lacP, and CAP binding site mutations were sequence verified. The sequencing quality for the lacI open reading frame, lacO, and the bla ribosome binding site was not high enough to conclude that the mutants were there. It was time to test!

![BlalacIP](https://static.igem.org/mediawiki/2021/0/0a/T--WLC-Milwaukee--blalacIP.png)
![BlalacI](https://static.igem.org/mediawiki/2021/b/b0/T--WLC-Milwaukee--blalacI.png)
![BlaCAP](https://static.igem.org/mediawiki/2021/f/f5/T--WLC-Milwaukee--blaCAP.png)
![BlalacP](https://static.igem.org/mediawiki/2021/7/78/T--WLC-Milwaukee--blalacP.png)
![BlalacO](https://static.igem.org/mediawiki/2021/b/bf/T--WLC-Milwaukee--blalacO.png)
![BlaRBS](https://static.igem.org/mediawiki/2021/9/90/T--WLC-Milwaukee--blaRBS.png)

![Test](https://static.igem.org/mediawiki/2021/1/1d/T--WLC-Milwaukee--test.png)

Our testing indicates that every mutant is functioning as expected! **For more information, see our [Demonstrate page.](https://2021.igem.org/Team%3AWLC-Milwaukee/Proof_Of_Concept)**

* [Project](%5CTeam%3AWLC-Milwaukee%5CDescription)
* [Description](%5CTeam%3AWLC-Milwaukee%5CDescription)
* [Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
* [Engineering](%5CTeam%3AWLC-Milwaukee%5CEngineering)
* [Design](%5CTeam%3AWLC-Milwaukee%5CDesign)
* [Results](%5CTeam%3AWLC-Milwaukee%5CResults)
* [Implementation](%5CTeam%3AWLC-Milwaukee%5CImplementation)
* [Lab Manual](%5CTeam%3AWLC-Milwaukee%5CCommunication)
* [Demonstrate](%5CTeam%3AWLC-Milwaukee%5CProof_Of_Concept)
* [Contribution](%5CTeam%3AWLC-Milwaukee%5CContribution)
* [Improve](%5CTeam%3AWLC-Milwaukee%5CImprove)

* Parts
* [Parts](%5CTeam%3AWLC-Milwaukee%5CParts)
* [Part Collection](%5CTeam%3AWLC-Milwaukee%5CPart_Collection)
* [Experiments](%5CTeam%3AWLC-Milwaukee%5CExperiments)

* [Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
* [Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
* [Integrated Human Practices](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices%5CiHP)
* [Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)
* [Education](%5CTeam%3AWLC-Milwaukee%5CEducation)

* [Team](%5CTeam%3AWLC-Milwaukee%5CTeam)
* [Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
* [Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)
* [Medals](%5CTeam%3AWLC-Milwaukee%5CMedals)
* [Awards](%5CTeam%3AWLC-Milwaukee%5CAwards)

![Wisconsin Lutheran College](https://static.igem.org/mediawiki/2018/7/79/T--WLC-Milwaukee--WLC.png)

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

# Team:WLC-Milwaukee/Experiments

Header

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2021/9/93/T--WLC-Milwaukee--biglogotrnsparent.png "WLC-Milwaukee iGEM Home")](/Team%3AWLC-Milwaukee)

[PROJECT](%5CTeam%3AWLC-Milwaukee%5CDescription)

[Description](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Engineering](%5CTeam%3AWLC-Milwaukee%5CEngineering)
[Design](%5CTeam%3AWLC-Milwaukee%5CDesign)
[Results](%5CTeam%3AWLC-Milwaukee%5CResults)
[Implementation](%5CTeam%3AWLC-Milwaukee%5CImplementation)
[Lab Manual](%5CTeam%3AWLC-Milwaukee%5CCommunication)
[Demonstrate](%5CTeam%3AWLC-Milwaukee%5CProof_Of_Concept)
[Contribution](%5CTeam%3AWLC-Milwaukee%5CContribution)
[Improve](%5CTeam%3AWLC-Milwaukee%5CImprove)

[PARTS](%5CTeam%3AWLC-Milwaukee%5CParts)

[Parts](%5CTeam%3AWLC-Milwaukee%5CParts)
[Part Collection](%5CTeam%3AWLC-Milwaukee%5CPart_Collection)
[Experiments](%5CTeam%3AWLC-Milwaukee%5CExperiments)

[HUMAN PRACTICES](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)

[Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Integrated Human Practices](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices%5CiHP)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)
[Education](%5CTeam%3AWLC-Milwaukee%5CEducation)

[TEAM](%5CTeam%3AWLC-Milwaukee%5CTeam)

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)
[Medals](%5CTeam%3AWLC-Milwaukee%5CMedals)
[Awards](%5CTeam%3AWLC-Milwaukee%5CAwards)

[![WLC-Milwaukee](https://static.igem.org/mediawiki/2021/9/93/T--WLC-Milwaukee--biglogotrnsparent.png "WLC-Milwaukee")WLC-Milwaukee](/Team%3AWLC-Milwaukee)

PROJECT

[Description](%5CTeam%3AWLC-Milwaukee%5CDescription)
[Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
[Engineering](%5CTeam%3AWLC-Milwaukee%5CEngineering)
[Design](%5CTeam%3AWLC-Milwaukee%5CDesign)
[Results](%5CTeam%3AWLC-Milwaukee%5CResults)
[Implementation](%5CTeam%3AWLC-Milwaukee%5CImplementation)
[Lab Manual](%5CTeam%3AWLC-Milwaukee%5CCommunication)
[Demonstrate](%5CTeam%3AWLC-Milwaukee%5CProof_Of_Concept)
[Contribution](%5CTeam%3AWLC-Milwaukee%5CContribution)
[Improve](%5CTeam%3AWLC-Milwaukee%5CImprove)

DOCUMENTATION

[Parts](%5CTeam%3AWLC-Milwaukee%5CParts)
[Part Collection](%5CTeam%3AWLC-Milwaukee%5CPart_Collection)
[Experiments](%5CTeam%3AWLC-Milwaukee%5CExperiments)

HUMAN PRACTICES

[Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
[Integrated Human Practices](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices%5CiHP)
[Collaborations](%5CTeam%3AWLC-Milwaukee%5CCollaborations)
[Education](%5CTeam%3AWLC-Milwaukee%5CEducation)

TEAM

[Members](%5CTeam%3AWLC-Milwaukee%5CTeam)
[Attributions](%5CTeam%3AWLC-Milwaukee%5CAttributions)
[Medals](%5CTeam%3AWLC-Milwaukee%5CMedals)
[Awards](%5CTeam%3AWLC-Milwaukee%5CAwards)

[![Instagram](https://static.igem.org/mediawiki/2021/c/c6/T--WLC-Milwaukee--instagram.png)](https://www.instagram.com/wlcigem/)
![Email](https://static.igem.org/mediawiki/2021/8/8f/T--WLC-Milwaukee--email.iconw.png)

/\*Make text red and bold\*/
.red\_text {
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}

WLC-Milwaukee 2021 | Experiment

Experiment

# Example Experiment

This project required many basic synthetic biology techniques. See below for the techniques we used, reagents required, and a brief protocol. Note that all techniques require basic microbiology tools (pipettes, tubes, plates, incubator, etc.)

1. Plasmid miniprep (IBI scientific plasmid miniprep kit)

a. Add 1.5ml culture to microcentrifuge tube
b. Centrifuge at 15,000 x g for 30s, discard flow through

i. Repeat until culture is gone
c. Resuspend pellet of cells in 200 ul PD1 buffer
d. Add 200 ul PD2 buffer, invert tube 10 times to mix, let stand 2 min
e. Add 300 ul PD3 buffer, invert tube 10 times to mix
f. Centrifuge at 15,000 x g for 3 min
g. Add supernatant to PD column with 2ml collection tube
h. Centrifuge at 15,000 x g for 30s, discard flow through
i. Add 400 ul W1 buffer to column
j. Centrifuge at 15,000 x g for 30s, discard flow through
k. Add 600 ul of wash buffer to column
l. Centrifuge at 15,000 x g for 30s, discard flow through
m. Centrifuge at 15,000 x g for 3 min, discard flow through
n. Place PD column into microcentrifuge tube
o. Add 30 uL of sterile water to column, let stand 2 min
p. Centrifuge at 15,000 x g for 2 min
q. Discard column, reserve microcentrifuge tube. Store at 4 C

2. PCR

a. Reagents
b. Protocol:

i. Aliquot into PCR tubes:

1. 9 ul sterile water
2. 12.5 ul mastermix
3. 1.25 ul rev primer
4. 1.25 ul forward primers
5. 1 ul template DNA

ii. Place in thermocycler at:

1. 95 C for 30s
2. 60 C for 30s
3. 72 C for 180s
4. Repeat 40x
5. 4 C hold until ready to use

3. RE digest

a. To microcentrifuge tube, add:

i. 10 ul DNA of interest
ii. 4 ul sterile water
iii. 4 ul buffer
iv. 1 ul of restriction enzyme

b. Incubate in a water bath at 37 C for 1 hr

4. Ligation

a. To PCR tube, add:

i. 6 ul DNA of interest
ii. 2 ul plasmid of interest
iii. 1 ul buffer
iv. 1 ul T4 DNA ligase

b. Incubate at 16 C overnight

5. Transformation

a. To microcentrifuge tube, add:

i. 50 ul DH5a electrocompetent cell
ii. 5 ul ligation

b. Add solution to electroporation cuvette
c. Pulse
d. Quickly add 1ml of LB broth to cuvette
e. Transfer solution from cuvette to microcentrifuge tube
f. Incubate at 37 C for 30 min
g. Centrifuge at 13.7 x g for 1 min
h. Decant LB
i. Add 50 ul of new LB broth, resuspend pellet
j. Spread on LB agar plate
k. Incubate plate overnight at 37 C

6. Gel electrophoresis

a. To 100 ml erlenmeyer flask, add:

i. 0.4g agarose
ii. 50ml TAE buffer

b. Heat flask, swirling occasionally until all agarose is dissolved and solution is clear
c. Add 2.5ul GelRed
d. Pour into gel cast with lane forks
e. Allow to solidify
f. Add 5ul DNA loading dye to DNA samples
g. Add 15 ul ladder to lane 1
h. Add 30 ul DNA sample to each lane
i. Run gel at 120V for 45 minutes
j. Visualize under UV light

7. Gel extraction (IBI scientific gel extraction protocol)

a. Excise gel fragments of interest and add to microcentrifuge tube
b. Add 500 ul of DF buffer and vortex for 15s
c. Incubate in water bath at 55 C until gel has dissolved
d. Place DF column in 2 ml collection tube, transfer 800 ul of solution to column
e. Centrifuge at 15,000 x g for 30s, discard flow through
f. Add 400 ul of W1 buffer to column
g. Centrifuge at 15,000 x g for 30s, discard flow through
h. Add 600 ul of wash buffer to column. Let stand 1 min
i. Centrifuge at 15,000 x g for 30s, discard flow through
j. Centrifuge at 15,000 x g for 3 min, discard flow through
k. Transfer column to new microcentrifuge tube
l. Add 30 ul sterile water to column. Let stand 2 min
m. Centrifuge at 15,000 x g for 2 min
n. Discard column and reserve microcentrifuge tube -- store at 4 C

8. Gibson assembly

a. Prep two PCR tubes. (Always use 10 uL of the Gibson assembly master mix and then 10 uL of the other components, including the sterile water)

i. One with 1 uL of pSB1C3, 1 uL of LacZ fragment, 1 uL of LacY fragment, 2 uL of LacI fragment, 5 uL of sterile water, 10 uL Gibson assembly master mix
ii. The other has 0.5 uL of pSB1C3, the LacZ fragment, and the LacY fragment, 1 uL of the LacI fragment, 7.5 uL of sterile water, and 10 uL of Gibson assembly master mix

b. Place in thermocycler for 1 hr (because of the 4 fragments) at 50 C
c. Hold at 4 C until ready for use
d. Proceed with Transformation Protocol

9. Nitrocefin Solubility

a. Find the desired E.coli culture concentration

i. Dilute a 1 mL E.coli ampicillin resistant culture

1. Transfer 100 uL of culture to microcentrifuge tube of 900 uL of LB broth
2. Transfer 100 uL of that culture to another microcentrifuge tube containing 900 uL of LB broth
3. Transfer 100 uL of previous culture to a final microcentrifuge tube containing 900 uL of LB broth

ii. Add 10 uL of 100% concentration of Nitrocefin into each of the dilutions
iii. Wait 30 minutes and observe any color change

b. Find the minimal concentration of Nitrocefin necessary

i. Dilute Nitrocefin to 50, 25, 12.5, and 6.25% of original (100mg/uL) concentration
ii. Add 10uL Nitrocefin to 1 mL E.coli cultures
iii. Observe any color change

10. Mutagenesis

a. Aliquot into PCR tubes:

i. 12.5 uL Q5 Hot Start High-Fidelity 2X Master Mix
ii. 1.25 uL 10uM Forward Primer
iii. 1.25 uL 10uM Reverse Primer
iv. 1 uL Template DNA
v. 9 uL sterile water

b. Place in thermocycler:

i. Lac I stop and Lac Z RBS mutation

1. 98 degrees C for 30 seconds
2. 98 degrees C for 10 seconds
3. 57 degrees C for 30 seconds
4. 72 degrees C for 2 minutes
5. Repeat steps 2-4 for 25 cycles
6. Hold for infinity at 4 degrees C

ii. Lac O and CAP deletion mutation

1. 98 degrees C for 30 seconds
2. 98 degrees C for 10 seconds
3. 60 degrees C for 30 seconds
4. 72 degrees C for 2 minutes
5. Repeat steps 2-4 for 25 cycles
6. Hold for infinity at 4 degrees C

iii. Lac P deletion and Lac I promoter mutation

1. 98 degrees C for 30 seconds
2. 98 degrees C for 10 seconds
3. 64 degrees C for 30 seconds
4. 72 degrees C for 2 minutes
5. Repeat steps 2-4 for 25 cycles
6. Hold for infinity at 4 degrees C

c. Perform KLD reaction

i. Add to tube:

1. 1 uL of PCR product
2. 5 uL 2X KLD Reaction Buffer
3. 1 uL 10X KLD Enzyme Mix
4. 3 uL sterile water

ii. Incubate at room temperature for 5 minutes

d. Perform Transformation

i. Add 5 uL of KLD mix to 50 uL of chemically-competent cells
ii. Incubate on ice for 30 minutes
iii. Heat shock at 42 degrees C for 30 seconds
iv. Incubate on ice for 5 minutes
v. Add 950 uL of SOC media
vi. Incubate for 1 hr at 37 degrees C
vii. Spread 100 uL onto LB chloramphenicol plates and incubate at 37 degrees C overnight

* [Project](%5CTeam%3AWLC-Milwaukee%5CDescription)
* [Description](%5CTeam%3AWLC-Milwaukee%5CDescription)
* [Safety](%5CTeam%3AWLC-Milwaukee%5CSafety)
* [Engineering](%5CTeam%3AWLC-Milwaukee%5CEngineering)
* [Design](%5CTeam%3AWLC-Milwaukee%5CDesign)
* [Results](%5CTeam%3AWLC-Milwaukee%5CResults)
* [Implementation](%5CTeam%3AWLC-Milwaukee%5CImplementation)
* [Lab Manual](%5CTeam%3AWLC-Milwaukee%5CCommunication)
* [Demonstrate](%5CTeam%3AWLC-Milwaukee%5CProof_Of_Concept)
* [Contribution](%5CTeam%3AWLC-Milwaukee%5CContribution)
* [Improve](%5CTeam%3AWLC-Milwaukee%5CImprove)

* Parts
* [Parts](%5CTeam%3AWLC-Milwaukee%5CParts)
* [Part Collection](%5CTeam%3AWLC-Milwaukee%5CPart_Collection)
* [Experiments](%5CTeam%3AWLC-Milwaukee%5CExperiments)

* [Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
* [Outreach & Engagement](%5CTeam%3AWLC-Milwaukee%5CHuman_Practices)
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# Team:WLC-Milwaukee/Results

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WLC-Milwaukee 2021 | Results

Results

# Overview

Successful results in the lab are integral to our laboratory kit and its implementation in the classroom. Despite a few hiccups, we were able to achieve results that show that our lab kit will perform and function as intended. A brief summary of our results in the laboratory can be found below. For a more in-depth look at our work in the lab, see our [Demonstrate page.](https://2021.igem.org/Team%3AWLC-Milwaukee/Proof_Of_Concept)

# Ideal Nitrocefin Concentration

Pictured below are the results from our initial nitrocefin concentration tests. Based on these tests, we were able to pinpoint the best concentration of nitrocefin and *E. coli* to demonstrate a significant color change in a reasonable amount of time. This allowed us to make modifications to our lab kit and manipulate our directions to ensure the students quickly got easy-to-visualize results.

![Nitro Results](https://static.igem.org/mediawiki/2021/1/1f/T--WLC-Milwaukee--nitroresults.png)

# Sugar Concentration

Pictured below are the results from testing 10% lactose and glucose over different incubation times. Every microcentrifuge tube contains our modified *E. coli* and nitrocefin. The concentration of nitrocefin in the left image is 10 ug/mL the right image is 1 ug/mL nitrocefin. The key takeaway from these tests was that after 30 minutes, there was a visible difference in the color change that occurred in the presence of lactose. Additionally, 1 ug/mL nitrocefin allowed for a visible gradient. These results were implemented into the instructions of the lab manual to optimize the time and resources available to teachers.

![Color Change](https://static.igem.org/mediawiki/2021/8/87/T--WLC-Milwaukee--colorchange.png)
![Sugar Results](https://static.igem.org/mediawiki/2021/2/2e/T--WLC-Milwaukee--sugarresults2.png)

# Mutagenesis results

We mutated six different regions within the lac operon with site-directed mutagenesis to bolster the applicability of our lab kit to higher levels of education. The six regions we mutated were lacO, the lacI promoter, lacP, the CAP binding site, the bla ribosomal binding site, and the lacI open reading frame. The mutations prevent the regions from functioning normally, allowing for the determination of what part of the operon is nonfunctional through observing the color change.

![blalinear](https://static.igem.org/mediawiki/2021/f/fd/T--WLC-Milwaukee--blalinear.png)

Pictured below is an example of one of the mutant's functions. The promoter sequence for bla, lacP, is inactivated. There is no color change despite nitrocefin being present in the solution. This is because expression is unable to occur without a promoter. Therefore, no b-lactamase is produced and no color change is observed. This is one example of how easy it is to deduce the function of individual genes within the lac operon with our lab kit!

![blalinear](https://static.igem.org/mediawiki/2021/0/09/T--WLC-Milwaukee--capmut.png)

# Future Research

Within synthetic biology and science in general, negative results can be just as important as positive results and can guide potential research projects in the future. Initially with our project, we wanted to use lacZ and its product b-galactosidase to turn a substrate, X-Gal, blue. However, due to the size of lacZ along with the regulatory components and lacY, we were unable to place the genes into the pSB1C3 plasmid. The gene we used, bla, works as intended and successfully demonstrates the function of the lac operon. However, traditional cloning was not sufficient and more research should be done with lacZ to determine if it can be inserted into a plasmid.

In searching for a substitute gene and substrate to use, we identified two alternates: bla and nitrocefin, and icm (indole-3-carboxylate monooxygenase) and indole 3 carboxylic acid. Unfortunately, icm and indole 3 carboxylic acid, which induces a purple color change, did not function. Due to time constraints, we did not explore the reasoning behind why the icm construct did not produce any results in the lab since we ended up using the bla construct. More investigation is necessary to find more options to demonstrate gene regulation in the laboratory instead of using lacZ.

For our site-directed mutagenesis results, we were only able to get verified sequencing for the lacI promoter, lacP, and the CAP binding site. While we were not able to conclude that mutations were present in the lacO, lacI open reading frame, and bla ribosome binding site, our results in the lab indicate that those three mutations are likely present. Still, it would be beneficial to obtain conclusive sequencing for those three mutageneses. In addition to this, our CAP binding site mutagenesis experiment did not produce results to the extent that we were expecting. We performed additional testing in order to explore this issue, but our results were subjective. To confirm our results from the CAP binding site mutagenesis experiment, spectrophotometry should be performed to quantify our results.

The purpose of our lab kit is to provide a reliable method of visualizing the functions of different genes within the lac operon. Results can be seen by the naked eye in the lab very quickly and are relatively cost-effective. However, it would be interesting to perform spectrophotometry using our kit to provide quantification of how well b-lactamase is cleaving nitrocefin in the different environments. As of right now, our iGEM team does not have a protocol for this. It would add depth and precision to our lab kit if we used spectrophotometry to measure the color change. Our team hopes to achieve this in the future.