iGEM 2018 — Team Duke

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# Team:Duke/Notebook

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PROJECT

[Description](https://2018.igem.org/Team%3ADuke/Description)
[Design](https://2018.igem.org/Team%3ADuke/Design)
[Experiments](https://2018.igem.org/Team%3ADuke/Experiments)
[Notebook](https://2018.igem.org/Team%3ADuke/Notebook)
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[Parts Overview](https://2018.igem.org/Team%3ADuke/Parts)
[Basic Parts](https://2018.igem.org/Team%3ADuke/Basic_Part)

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[Human Practices](https://2018.igem.org/Team%3ADuke/Human_Practices)

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[Education & Engagement](https://2018.igem.org/Team%3ADuke/Public_Engagement)
[Model](https://2018.igem.org/Team%3ADuke/Model)

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

# Notebook

## Cloning: Daniel

## Enzyme Assays: Joe

## Promoter Characterization: Maria


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# Team:Duke/Experiments

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[Description](https://2018.igem.org/Team%3ADuke/Description)
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[Basic Parts](https://2018.igem.org/Team%3ADuke/Basic_Part)

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[Education & Engagement](https://2018.igem.org/Team%3ADuke/Public_Engagement)
[Model](https://2018.igem.org/Team%3ADuke/Model)

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

# Experiment

# Protocols

Below is a document containing all the lab protocols written during experiments for this project. Please see the notebook page for further elaboration on what steps were taken when and by whom. Parts used in experiments not previously documented in the Registry have been added to or updated in the Registry this year.

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# Team:Duke/Results

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[Description](https://2018.igem.org/Team%3ADuke/Description)
[Design](https://2018.igem.org/Team%3ADuke/Design)
[Experiments](https://2018.igem.org/Team%3ADuke/Experiments)
[Notebook](https://2018.igem.org/Team%3ADuke/Notebook)
[Model](https://2018.igem.org/Team%3ADuke/Model)
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[Attributions](https://2018.igem.org/Team%3ADuke/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ADuke/Parts)
[Basic Parts](https://2018.igem.org/Team%3ADuke/Basic_Part)

[SAFETY](https://2018.igem.org/Team%3ADuke/Safety)
[Human Practices](https://2018.igem.org/Team%3ADuke/Human_Practices)

AWARDS

[Education & Engagement](https://2018.igem.org/Team%3ADuke/Public_Engagement)
[Model](https://2018.igem.org/Team%3ADuke/Model)

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

# Protein Expression Results

To test the expression levels of our proteins, we used the shake flask protein expression technique. The specific details of the protocol can be found in the protocol section of the website.
Prior to performing the SDS-page, we made sure that all the OD600 for our cells (BAPT, DBAT, badA, tax10, tycA) are around 10.0 for consistency of protein amount in SDS-page.
After the SDS-page has finished running, the gel is stained and destained and a greyscale image of the gel look like the follows:

As seen from the graph, from the right to the left, the lanes are the ladder (Mark12TM Unstained Standard), BAPT, DBAT, badA, tax10, and tycA). Although the gel was broken during the destaining process, we were able to put the gel back together. To demonstrate the most accurate level of protein expression, we changed the contrast of the image so that only dark bands on the gel can be seen when using the imaging software, the contrasted image is shown below:

And the ladder we used have the following configuration:

We then opened the software imageJ, and highlighted the lane of the ladder to be the reference and selected each lane from left to right so that imageJ can automatically determine the level of concentration in a certain location of the gel, and the normalized result is shown below:

As seen from above, there are 6 panels in the graph, corresponding to (ladder, BAPT, DBAT, badA, tax10, and tycA), and from the left to the right of each panel, the expression levels correspond to from bigger size proteins to smaller size proteins. By using the molecular sizes of proteins and the ladder, we are able to isolate the peaks that correspond to BAPT, DBAT, badA, tax10, and tycA, which are shown in the graph by drawing 2 straight lines from the base of the peak to the bottom of the panel. Therefore, we can calculate the expression levels of proteins in a gel by dividing the area of the peak by the entire area. The protein expression levels for BAPT, DBAT, badA, tax10 are 19.8%, 21.3%, 20.5%,16.7%, respectively. Because we cannot find the protein band that corresponds to the tycA molecular mass, we could not determine protein expression in tycA in this SDS-gel page experiment. The results were repeated in different gels to determine if tycA expression level can be determined, but the result proved to be inconclusive. One possible explanation could be that during the denaturation of the cells, tycA could disintegrate into its different domains due to size, and the protein is thus separated into segments and could not express as the entire enzyme. More work needs to be done to determine protein expression for tycA.