Team:LambertGA/Results


Results

Inoculations of GFP Constructs (58 tubes)

After making our constructs, we inoculated cultures from previous transformations that have successfully expressed the fluorescent protein.

1.P-Lambda-R--LacI--GFP (no deg tag)
  1. 3 tubes in each cell type (DH10, Keio Wild, Keio ClpP) induced with 0 uM IPTG (plain LB)
  2. 3 tubes of each cell type (DH10, Keio Wild, Keio ClpP) induced with 100 uM IPTG (plain LB)
  3. RESULTS:
2. P-Lambda-R--LacI--GFP (DAS)
  1. 3 tubes in each cell type (DH10, Keio Wild, Keio ClpP) induced with 0 uM IPTG (plain LB)
  2. 3 tubes of each cell type (DH10, Keio Wild, Keio ClpP) induced with 100 uM IPTG (plain LB)
  3. RESULTS:
3. P-Lambda-R--LacI--GFP (LAA)
  1. 3 tubes in each cell type (DH10, Keio Wild, Keio ClpP) induced with 0 uM IPTG (plain LB)
  2. 3 tubes of each cell type (DH10, Keio Wild, Keio ClpP) induced with 100 uM IPTG (plain LB)
  3. RESULTS:
4. Plain LB, DH10 cells in plain LB, Keio Wild cells in plain LB, and Keio ClpP cells in plain LB
5. RESULTS:
  1. 10/17: The GFP constructs were brought to the plate reader at Georgia Tech. Although cells were grown in the liquid culture, they did not fluoresce.
6. Pictures:

Data from the Plate Reader


All of the GFP constructs as liquid cultures.


Julia Leveille and Lauren Hong with the 58 liquid cultures before analyzing them on the plate reader at Georgia Tech.


Troubleshooting for Lack of Proper Tube Labeling


We plated our constructs in all three cell types(DH10, Keio Wild, and Keio ClpP) on Kanamycin, Tetracycline, Chloramphenicol, and Ampicillin. We were testing to verify what backbones the plasmids were in. As the image shows, our cells grew in most of the plates resistant to the specific antibiotics. We are in the process of figuring out how we obtained these results, but we hypothesized that our cells contain constructs with all the vectors with backbones resistant to those antibiotics.




Sequencing Results



The R0011 ClpX part of our DNA construct was sent in for sequencing and confirmed to be matching DNA through Eurofins MWG Operon.


The ClpP B0033 CI part of our DNA construct was also sent in for sequencing and confirmed to be matching DNA through Eufofins MWG Operon.



Expected Results


  1. Keio Wild Strains: In the Keio Wild Strains, we assembled two constructs - one with a degradation tag, and one without a degradation tag. Since ClpXP can only degrade proteins that have been tagged, we expect our plates with the degrons to have a lower concentration of purple cells than the plates without the degrons. The following graph shows the relationship between time and expected concentration of purple cells for the Keio Wild strain; “T” represents the time at which the cells were induced by IPTG.



  2. Keio ClpX Knockout Strain: In this strain, ClpX will be knocked out and ClpP will be allowed to function. ClpX is responsible for de-linearizing the target protein into its primary structure and then translocating it into a proteolytic cavity in ClpP. Since ClpX will not be expressed, the chromoprotein will not be de-linearized and hence prevent ClpP from degrading the primary structure of the protein. With this in mind, we expect our plates to remain purple in the Keio ClpX Knockout strain.
    The following graph shows the relationship between time and expected concentration of purple cells for the Keio ClpX Knockout strain; “T” represents the time at which the cells were induced by IPTG.



  3. Keio ClpP Knockout Strain: In this strain, ClpP will be knocked out and ClpX will be allowed to function. ClpP is responsible for degrading the primary structure of the protein itself; it does this by breaking the individual covalent bonds (polypeptide bonds) that exist between the amino acids in the polypeptide chain. Since ClpP will not be expressed, the chromoprotein will not be degraded into individual amino acids. However, despite this, the chromoprotein will still be degraded partially because it will be de-linearized by ClpX. In other words, although the protein will still be in its primary structure, it will still lack the hydrogen bonds, hydrophobic interactions, ionic bonds, disulfide bridges, and R-Group interactions that exist in its tertiary (or in some cases, quaternary) structure; consequently, that will limit the chromoprotein’s ability to express its pigments. With this in mind, we expect our plates to have a smaller number of purple cells present in the Keio ClpP Knockout strain.
    The following graph shows the relationship between time and expected concentration of purple cells for the Keio ClpP Knockout strain; “T” represents the time at which the cells were induced by IPTG.



    Medals




    Bronze
    Team formed and registered
    Wiki completed
    Poster completed
    Presentation ready for Jamboree
    Attributed all work done for project
    Part (BBa_K1911001) documented and submitted
    All forms submitted


    Silver
    New part (BBa_K1911000) documented, submitted, and validated
    Collaboration: iGEM teams of Alverno California and CAPS_Kansas and Georgia State University (GSU), the Styczynski Lab from the Georgia Institute of Technology (GaTech), Centers for Disease Control and Prevention (CDC), New England Biolabs, and Lambert High School’s engineering department
    Human Practices: Maker Faire, Atlanta Science Festival (ASF), Regional Education Service Agencies (RESA), Next Generation Focus (NGF), Congenital Heart Defect Walk, Sharon Science Days, Micronutrient Discussion with the CDC, collaboration with the Styczynski Lab from GaTech


    Gold
    Integrated Human Practices: collaboration with Styczynski Lab at GaTech with real-world applications of improving the zinc biosensor for malnourished and micronutrient-deficient countries
    Improved previous iGEM project parts: BBa_K1033906, BBa_M0050-LAA, BBa_M0052-DAS
    Proof of Concept: GFP analysis