Team:Duke/Results

DESIGN STATEMENT

To design and express a significantly more thermostable Griffithsin dimer and monomer for future implementation in a diagnostic assay.

MODELING of GRIFFITHSIN

Our design began with taking the originally published sequence of griffithsin and modeling thermostable changes in the sequence. Click here for more information on our modeling.

DESIGNING A HIGH-EXPRESSING CONSTRUCTS

After creating an optimal amino acids sequence from the Yasara modeling, we codon optimized the sequence in E. coli via

IPTG INDUCED EXPRESSION

To mimic industrial expression protocols, we created a protocol to first maximize the biomass of the Griffithsin-expressing cells. Then the IPTG was added to turn on the promoter and hijack the cell’s metabolism to producing GRFT. The GRFT was then removed from the cells.

To express griffithsin (GRFT) at maximum levels, dynamic metabolic control was exercised using IPTG induction as the metabolic valve.

  1. Biomass is grown in of SM10++(max 10g/L) media for 18 hours
  2. IPTG rich SM0++ media is then added to the culture which induces:
    1. The vast majority of its metabolism shifts to protein creation as the strong promoter is turned on.
    2. Cell growth will dramatically decrease
  3. Homgenization
    1. Since Griffithsin is an intracellular protein, homogenization was chosen as the method taken to lyse the cells.
    2. Homogenization feeds a sample through small aperture where the cell is lysed by shearing

Figure: Homogenization

CONFIRMATION OF GRIFFITHSIN EXPRESSION

We ran an SDS page gel on the lysate to confirm expression of griffithsin. The protocol we used for these is in the experiments tag but it is also diagramed below.

The following gel shows the presence of griffithsin.

Figure 1: 12% Bis-Tris SDS-PAGE. Lanes: NEB 10-200 kDa Ladder Ladder (L), Empty pSmart Vector (EV), Thermostable Griffithsin Monomer (TSm), Thermostable Griffithsin Dimer (TS)

Hydropathicity Considerations

Previous studies in our lab produced the following SDS-PAGE gel confirming that Wildtype-Griffithsin runs at 12 kDa.

Figure 2: From a previous experiment.


However, our SDS-Page gels produced a band at ~35 kDa. We hypothesize that the higher band results from the increased hydrophobicity from the thermostability modifications. The hydrophobicity significantly impacts the electrophoresis of proteins because the more hydrophobic the protein is, the more SDS binds to it (http://www.pnas.org/content/106/6/1760.full). Since SDS is negatively charged, additional binding would significantly reduce the electromagnetic driving force of GRFT, causing it to run higher.

Figure 3: Thermostability