iGEM 2021 — Team Baltimore_BioCrew

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

# Team:Baltimore BioCrew/Notebook

2021 Baltimore Biocrew

![Notebook Header](https://static.igem.org/mediawiki/2021/2/2f/T--Baltimore_BioCrew--Header_Notebook.pdf)

# Notebooks

## Contribution

## Engineering a Part

A project by the Baltimore BioCrew 2021 team.

##### Contact

*mail*
info@bugssonline.org

Baltimore BioCrew 2021

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

# Team:Baltimore BioCrew/Engineering

2021 Baltimore Biocrew

![Safety Header](https://static.igem.org/mediawiki/2021/e/eb/T--Baltimore_BioCrew--eng.png)

# Engineering

We introduced genes that affect mental health and mood into E. coli with the ultimate goal of putting them into a Lactobacillus probiotic strain. Click each section to learn more!

* Design

  We selected three genes that each affect brain function (click on gene name for a link to the registry page):
  + [TPH1:](http://parts.igem.org/Part%3ABBa_K3851001) The TPH1 gene in humans uses L-tryptophan to produce 5-HTP, the biochemical precursor to serotonin. Serotonin reduces the effects of depression and anxiety.
  + [ChAT:](http://parts.igem.org/Part%3ABBa_K3851003) The ChAT gene catalyzes the synthesis of acetylcholine in human cells. Acetylcholine is a neurotransmitter that works in the vagus nerve which connects the brain to the stomach.
  + [GAD:](http://parts.igem.org/Part%3ABBa_K3851004) GAD catalyzes the synthesis of GABA in human cells. GABA has been experimentally shown to reduce stress and is already used clinically to treat insomnia and depression.
  We are trying to combine these genes in Lactobacillus to make a psychobiotic strain that can improve people's mental health.

![](https://static.igem.org/mediawiki/2020/e/e5/T--Baltimore_BioCrew--MicroTubes.jpeg)

* Build
  *arrow\_downward*

  The steps that we used to design our parts was:
  + Identified different human genes involved in regulating mood and mental health
  + Found the genes from previous use in the iGEM Parts Registry.
  + Put each amino acid sequence into a standard NCBI protein BLAST search to confirm the gene.”
  - Added additional regulatory sequences by using a promoter, RBS, and terminator that had been used frequently before
  * Promoter: T7 promoter I719005
  * RBS: B0034
  * Terminator: B0015- Ordered genes as gBlocks from IDT
  - Digested gBlocks and pSB1C3 vector(with RFP gene insert) with EcoRI and PstI (protocols can be found at our  [Experiments](https://2021.igem.org/Team%3ABaltimore_BioCrew/Experiments) page.- Ligated vector and insert together and transformed into *E. coli*
    - Found *E.coli* that had successfully transformed, as indicated by absence of RFP
    - Picked out colonies and performed colony PCRWhile we obtained pink colonies for the positive control and colonies on the plates with our genes, we found that they all had the same sized insert, and that this insert was the wrong size.
  ![positive control](https://static.igem.org/mediawiki/2021/7/78/T--Baltimore_BioCrew--pos.png)
  ![genes](https://static.igem.org/mediawiki/2021/9/98/T--Baltimore_BioCrew--genes.png)

* Learn
  *arrow\_downward*

  We got into the lab late and thus only had time for one set of genes. After digest, ligation, transformation, and culture, our goal was to isolate a positive clone from any of the successful cultures of our genes in *E. coli,* which we chose to use to clone our plasmids for future use in Lactobacillus. After several iterations of colony screening, we did not get positive inserts. We therefore decided that something must be wrong with our cloning strategy and tried a different assembly method.

* Improve
  *arrow\_downward*

  The next assembly method that we tried was Gibson assembly by PCR amplifying our genes and inserts so that they had 20 bp of overlap. We obtained good products for our vector (left lanes) and our genes (right lanes). We then used the New England Biolabs HiFi DNA assembly method.

  ![](https://static.igem.org/mediawiki/2021/6/66/T--Baltimore_BioCrew--genes1.png)

  In the future, we want to test our genes in Lactobacillus. We would like to determine the growth rates of each modified strain and compare it to the many control samples we have already taken.

### Results

After several trials of cloning, we again obtained colonies indicating that our new cloning method was successful.

![](https://static.igem.org/mediawiki/2021/7/77/T--Baltimore_BioCrew--chat.png)

A project by the Baltimore BioCrew 2021 team.

##### Contact

*mail*
info@bugssonline.org

Baltimore BioCrew 2021

*swap\_vert*

* ###### SECTIONS
* [Back To Top](#home)

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

# Team:Baltimore BioCrew/Experiments

2021 Baltimore Biocrew

![Experiments Header](https://static.igem.org/mediawiki/2021/6/6c/T--Baltimore_BioCrew--Experiments_Header.jpg)

# Experiments

## Protocols

#### [Bacterial Transformation](https://static.igem.org/mediawiki/2021/f/fe/T--Baltimore_BioCrew--Protocols_Bacterial_Transformation.pdf)

#### [Gibson Assembly](https://static.igem.org/mediawiki/2021/4/4f/T--Baltimore_BioCrew--Protocols_Gibson_Assembly.pdf)

#### [Gel Electrophoresis](https://static.igem.org/mediawiki/2021/2/2c/T--Baltimore_BioCrew--Protocols_Gel_Electrophoresis.pdf)

#### [Ligation](https://static.igem.org/mediawiki/2021/8/8d/T--Baltimore_BioCrew--Protocols_Ligation.pdf)

#### [Restriction Digestion](https://static.igem.org/mediawiki/2021/d/d0/T--Baltimore_BioCrew--Protocols_Restriction_Digestion.pdf)

#### [SDS PAGE Gel](https://static.igem.org/mediawiki/2021/a/a4/T--Baltimore_BioCrew--Protocols_SDS_PAGE.pdf)

A project by the Baltimore BioCrew 2021 team.

##### Contact

*mail*
info@bugssonline.org

Baltimore BioCrew 2021

*swap\_vert*

* ###### SECTIONS
* [Back To Top](#home)

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

# Team:Baltimore BioCrew/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)