Results

Microscopy

We performed microscopy to measure the fluorescence per cell. This was done with samples that were incubated at 42 degrees for 12 hours. From these images, we noticed that TlpA and TcI worked only in some cells yet produced a very vibrant fluorescence. These were the heat-sensitive dimers. Alternatively, RpoH, Clp, HtpG, DnaK, GrpE, and LacIq are heat sensitive promoters. These produced more consistent fluorescence yet were not as bright. TlpA-NF contains a nonfluorescent mWasabi protein and thus, visualizing in the composite channel, produced the green background. No fluorescence was observed with this protein.

Further experiments may include computational analysis of fluorescence per cell and comparison to the different heat-sensitive strains to find the optimal mechanism for heating due to MNP excitation. Also, further microscopy using strains containing constitutive red fluorescence (mTangerine) to compare relative fluorescent protein expression.

A. TlpA

B. TlpA-NF

C. TcI

D. RpoH

E. HtpG

F. Clp

G. DnaK

H. GrpE

I. LacIq

Figure 1. Microscopy Images of 12-hour thermal induction of heat sensitive parts visualized using mWasabi expression: A-I: excitation and emission of mWasabi (green) and viewed using composite channel.

mWasabi/mTangerine Thermal Assay

We performed 7-hour thermal inductions of our heat-sensitive constructs and analyzed the fluorescence using a plate reader. We measured the results in triplicate and calculated standard deviation and standard error for the measurements. We performed these experiments with cells that expressed mWasabi through heat-sensitive promoters and a separate constitutive mTangerine. This allows comparison of mWasabi output due to different temperatures. The graphs are calculated using fluorescence divided by OD to give an approximate fluorescence per cell.

Further directions include repeating the assay with thermal induction via AMF stimulation of MNPs. Also, including samples that were not exposed to heat to compare any potential leaky fluorescence output. Furthermore, I would like to take measurements at various different time points to measure rate of expression of the system and optimal thermal induction time.

A

B

C

Figure 2. 7-hour thermal incubation at 40 C of heat sensitive constructs (mWasabi) in cells that constitutively express mTangerine. Fluorescence measured by plate reader normalized using cell density (OD600) to give approximate fluorescence per cell. Error bars gives standard error. A. Heat sensitive promoter mWasabi expression fluorescence measurements. B. Constitutive mTangerine expression fluorescence measurements. C. Combined graph of mWasabi and mTangerine expression for comparison.

A

B

C

Figure 3. 7-hour thermal incubation at 40 C of heat sensitive constructs (mWasabi) in cells that constitutively express mTangerine. Fluorescence measured by plate reader normalized using cell density (OD600) to give approximate fluorescence per cell. Error bars gives standard error. A. Heat sensitive promoter mWasabi expression fluorescence measurements. B. Constitutive mTangerine expression fluorescence measurements. C. Combined graph of mWasabi and mTangerine expression for comparison.

SDS-PAGE Gel

As another method of confirming that our protein was expressed, we ran our samples on a SDS-PAGE protein gel and stained it with Coomassie blue to confirm the presence of the mWasabi protein. Because the mWasabi expression isn’t able to be seen with the naked eye, this gel was used as a preliminary confirmation.

Future directions include running another SDS-PAGE gel after induction via AMF stimulation of MNPs. Also, running the gel with pre- and post- thermal induction of the samples to conclude that the band (and protein) appear in response to heat.

Figure 4. Coomassie stained SDS-PAGE gel of post thermal induction heat sensitive constructs. Band observed at 26.9 kDa confirms that mWasabi was produced due to the thermal induction.

Gradient Thermal Assay

We performed thermal induction assays with varying temperatures and culture agitations. We ran samples at 3 different temperatures, 37 C, 40 C, and 45 C. The cultures were either grown with shaking or in PCR tubes in a thermocycler. All the measurements are fluorescence normalized using OD/600 to determine relative fluorescence per cell. We observed increased expression of mWasabi with higher temperatures. Also, shaking tended to improve mWasabi expression, which would indicate that MNP attachment would be effective as it would be heating up the cells and not the media. There was no expression of mWasabi in TlpA-NF which is to be expected as this contains a non-fluorescent mWasabi.

Figure 2. Thermal Induction of heat sensitive constructs for 7-hours at varying time points. Blue: grown at 45 C in a larger culture while shaking. Orange, Grey, Yellow: grown at 37 C, 40 C, 45 C, respectively in separate 100 uL aliquots in a Thermocycler without shaking.

Amine-functionalized MNPs (AF-MNPs) binding to E.coli Surface

We tested for the OD of bacteria suspended in PBS before and after AF-MNPs were added to the solution. We used PBS instead of LB to decrease the number of other surfaces, such as proteins, to which the AF-MNPs can non-specifically bind to. After incubating the bacteria with AF-MNPs, the resulting nanoparticle-bacteria conjugate was removed with a magnet. Since the OD of the solution decreased with AF-MNPs, we have some evidence that suggests that the AF-MNPs were binding to the bacteria.

Feature direction includes running a TEM imaging on the nanoparticle-bacteria conjugate to confirm that the AF-MNPs are localizing around the bacterial surface. Also, we plan to test the binding capabilities of other types of MNPs, such as ones attached to antibodies.

Table 1. OD change before and after addition of MNPs

	Initial OD600 (no MNP added)	Final OD600 (MNP added and removed)
DH5alpha	Bba_J36848	LB + no antibiotic
Sample 1	7.006	5.874
Sample 2	1.886	0.561
Sample 3	0.411	0.608
Sample 4 (no cells, control)	0.202 (blank)	0.209 (blank)

Team:Pittsburgh/Results