Team:Stony Brook/Results

THE BUILDING PROCESS

We used a GST plasmid (GST-C-His +) so that melittin could remain in its inactive form when attached to the GST tag. A TEV cleavage site was included in between the GST protein and the melittin peptide sequence so that upon exogenous addition of TEV protease, melittin will be cleaved from GST, thus activating the protein.

In order to find the gene within the originating organism, we obtained samples of Apis mellifera, amd performed an RNA extraction of the venom gland of the honeybee. After using reverse transcriptase to get the cDNA library, we used PCR to search the cDNA library for prepromelittin precursor to melittin.

After multiple cDNA searches, we found the correct sized band for prepromelittin for 4 out of 6 of our samples. Once we saw we had the correct ~253 band of prepromelittin, we performed a gel extraction and used the DNA for another PCR.

We used this second PCR to add the TEV cleavage site sequence and amplify out the melittin portion, excluding the pre- and pro- precursor sequences. Once we saw the correct band of ~100, we were ready to ligate our insert into the digested GST plasmid.

Next, we wanted to induce our system with IPTG lac promoter to see if we could produce melittin, and so we grew out a culture, induced it, and performed SDS page alongside just the original GST plasmid to see if a size of ~30kDa would show up on the gel, which it did!

TESTING THE BIOACTIVITY OF MELITTIN

TESTING THE RESPONSE OF OUR RHLR BIOSENSOR

The initial testing of the 2011 Northwestern construct. 50uM of C4-HSL was added along with a negative control and GFP fluorescence was tracked over a period of seven hours. The A and B refer to two separate colonies that were picked off the plate. Everything worked as expected during log phase. However, once the negative control began approaching saturation, it began expressing GFP indicating that it may actually be a leaky promoter. This is useful to know so that once our system is complete, we know that we need to have the cells in log or lag phase to avoid false positives.

The data here is showing that GFP is being expressed after approximately 120 to 140 minutes in our initial test when no amount of C4-HSL was added

This growth curve is showing that fluorescence is being detected in the presence of C4-HSL when the cells are in log phase. However, it is also showing that the cells which did not receive the C4-HSL treatment are expressing GFP when they reach saturation.

This graph is showing that our cells are responding to the C4-HSL treatment. These cells were cotransfected with two plasmids. One plasmid has the Rhl promoter with mCherry acting as a reporter. The otherplasmid has our RhlR protein generator constitutively expressed.

The Northwestern was tested once again and we still saw the same results: GFP was expressed once cells reached saturation. Despite the low level of expression, it can still lead to false positives in the future.

To test to see if the Rhl promoter is in fact a leaky promoter, cells were transfected with plasmids containing just the Rhl promoter and mCherry. The results show that once cells reach saturation, fluorescence is detected, suggesting that Rhl is in fact a leaky promoter.

MATHEMATICAL MODELING

Legend:

Ae= Extracellular AHL

Ai= Intracellular AHL

d = AHL diffusion rate

g = decay rate of intracellular AHL

R= RhlR

r = RhlR synthesis rate

? = RhlR decay rate

C= AHL: RhlR complex (an active transcription factor)

b = binding rate of AHL with RhlR

� = dissociation rate of AHL:RhlR complex

G = Melittin DNA

Cg= AHL + RhlR + Melittin DNA complex

Kon = binding rate of the AHL:RhlR complex to the Melittin DNA

Koff = unbinding rate of AHL:RhlR complex from the Melittin DNA

M = Melittin mRNA

t = transcription rate for melittin

m = Melittin mRNA decay rate

P = Melittin Protein

T = translation rate for melittin

z = Protein Melittin�s decay rate

Pseudomonas aeruginosa produces AHLe (external C4HSL) and we assumed it is at a constant rate. Only part of AHLe diffuses into the membrane AHLi (internal C4HSL). AHL has a higher concentration out of the cell than inside. Due to the chemical driving force, there is a fast increase in diffusion of AHL into the cell. AHL eventually reaches chemical equilibrium and the AHLi remains relatively constant. The cell produces RhlR, but RhlR binds with AHL to form an AHLi-RHL complex, hence the amount of �free� RhlR decreases in the cell as it is forming the complex. The AHLi-RHL complex activates the transcription of melittin, hence increases the presence of melittin mRNA (MELT_mRNA). The melittin mRNA barely decreases over time because the degradation rate is slow and melittin mRNA is converted into protein melittin without being broken down. Melittin protein is produced at a fast rate and eventually the rate of production becomes constant.