Team:Baltimore BioCrew/Engineering

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: 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: 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: 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.

• 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 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 PCR
  While 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.

• 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.
  
  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.