Results

Inoculations of GFP Constructs (29 tubes)

1.In Keio Wild:

1. pλR LacI GFP Θ (no IPTG, 10µM IPTG, and 100µM IPTG) in 1C3
2. pλR LacI GFP DAS (no IPTG, 10µM IPTG, and 100µM IPTG) in 1C3
3. pλR LacI GFP LAA (no IPTG, 10µM IPTG, and 100µM IPTG) in 1C3

2. Controls

1. Plain Luria Broth
2. Keio Wild cells in plain Luria Broth

Colonies were chosen from the overnight plates and were diluted into 300 µL ultrapure water. These dilutions were used to inoculate triplicates of 4 milliliter cultures and were grown at 170 rpm for 24 hours in 37C. Each construct was treated to three different levels of IPTG induction(no IPTG, 10µM IPTG, and 100µM IPTG) to turn on the ClpXP degradation system and subsequently turn off GFP production. Our initial results were promising.

The constructs of the GFP with three different degradation tags were transformed into Keio Wild Type cells. Colonies were chosen from the overnight plates and were diluted into 300uL ultrapure water. These dilutions were used to inoculate triplicates of 4 milliliter cultures and were grown at 170 rpm for 24 hours in 37C. Each construct was treated to three different levels of IPTG induction to turn on the ClpXP degradation system and subsequently turn off GFP production. Our initial results were promising.

The graph (below) shows significant differences between the levels of induction and the resulting degradation of GFP. Of interest is the drop from 6000 GFP/OD for the GFP with no tag to the 2700 GFP/OD in the GFP LAA construct under 100uM IPTG induction. Our next step is to complete this experiment in parallel in both Keio ClpX knock out and ClpP knock out strains.


Our preliminary results demonstrated that our “Switch” construct worked. Under IPTG induction the levels of GFP were reduced in the Keio Wild strain.


The optimal densities of the Keio Wild cells from the inoculations.

Pictures:

Data from the Plate Reader

All of the GFP constructs as liquid cultures.

Julia Leveille and Lauren Hong with the 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