Enzymatic Pathway

Through the summer, we successfully constructed the tagged violacein pathway enzymes VioA, VioB, and VioE and submitted those parts to the registry. These three enzymes in series are capable of converting L-tryptophan into prodeoxyviolacein, a dark green colored intermediate of the violacein pathway. Each enzyme sequence was constructed to have the Avi-tag sequence included on its C-terminus, followed by the stop codon so that one single fusion protein is expressed. The avitag is capable of biotinylating the enzymes so that they can be bound to surfaces using a simple NeutrAvidin ligand system.

To simplify the construction process, each enzyme sequence was PCR'd off the iGEM Distribution sample with a KpnI site followed by the iGEM prefix, the gene of interest, and ended with an SphI or HindIII site that replaced the location of the stop codon. These PCR products could then be ligated into a modified pZE12-SMCS backbone which contained the Avitag sequence followed by the stop codon, iGEM suffix and ClaI site. This backbone was constructed using the primer insert method outlined in the Project Description.

With the modified enzymes, we performed experiments to show their expression and utilization of tryptophan using a UV/Vis spectrometer at the peak wavelengths at which tryptophan responds to (590,638, and 562 nm). The OD measurements of solutions contain equivolume of VioA, VioB, 40 mM Hydrogen Peroxide (for oxidation of tryptophan) and VioE lysate with varying concentration of tryptophan. The results are shown below.

The green points represent readings at 562 nm, blue at 590 nm, and red at 638 nm at 0.008 g/mL concentration of L-Tryptophan and 40 mM H₂0₂. As you can see, the absorbance of tryptophan noticably decreases over a time course of 1 hour. This experiment was recreated varying the ratio of enzymes and concentration of initial tryptophan. The results showed a similar decreasing trend.

This is the control tryptophan readings. In the absence of violacein enzymes, the amount of tryptophan showed the opposite trend.

Microfluidics Assembly

Binding Experiment

Goal - to determine if the biotinylated enzymes would attach to the streptavidin coated wall.

Experiment

• Negative Control - chip that is not coated with strepavidin through which we flowed ATTO 590.
• Test - streptavidin coated chip through which we flowed ATTO 590.
• Flow Rate - 5 uL/min
• Run Time - 20 min

After the experiment, air was flown through the chips and pictures were taken.

Results

The red fluorescence indicates that the enzyme successfully bound to the channel wall.

Control                              Test

Lysate Experiment

Goal - to determine the effect lysate would have on the binding efficiency between the streptavidin coated walls and the biotinylated enzymes.

Experiment

• Positive Control - streptavidin coated chip through which we flowed ATTO 520
• Negative Control - uncoated chip through which we flowed ATTO 520
• Test - streptavidin coated chip through which we flowed a 5:1 ratio of lysate to ATTO fdafdsfdfdsa 520.
• Flow Rate = 5 uL/min
• Total Run Time = 30 min

Entire setup was covered in aluminum foil and the lights were dimmed.

After the experiment, air was flown through the chips and pictures were taken. Channels were filled with DI water and stored in the 4 degree fridge wrapped in Al foil.

Results

The test chip showed a lower level of fluorescence compared to the positive control chip. This indicates that lysate has a small inhibitory effect on the binding between streptavidin and biotin.

Negative Control                   Positive Control               Test

Continuous Flow Experiment

Goal - to determine how long the attached enzymes would stay attached under continuous flow.

• Negative Control - noncoated chip
• Test - Flow DI water through a chip with ATTO 520 attached to the strepavidin coated PDMS for 15min.
• Flow Rate = 200 uL/min
• Total Run Time = 45 min, 3 15 min intervals

After each interval, the chip was washed with air and a picture was taken in air.

Results

The pictures indicate that the fluorescence gradually reduces under continuous flow. This suggests that the chips might have to be recoated after extended wear.

Control                               After 15min                   After 30min                        After 45min

GFP biobrick

Goal - to determine if our Avi tagged GFP will bind to our streptavidin coated chip.

Experiment

• Negative Control - A coated chip that is flushed with DI Water
• Test - A chip coated with streptavidin with filtered GFP lysate flown through
• Flow Rate - 5 uL/min
• Total Run Time - 20 min

The GFP containing cells were lysed with Bugbuster and filtered through a PD-10 Desalting Columns to remove excess biotin. Afterwards the chip was stored in DI Water in the fridge in aluminum foil. 4 days later we took the chip out and imaged it again. Then DI Water was flushed through and the chip was reimaged. It was seen that most of the binding occurred at the inlet port and the first channel. By the second channel there was no fluorescence found.

Results

The presence of green fluorescence shows that our biobrick successfully bound to the channel wall. Furthermore, green fluorescence was still present even after the chip was stored for 4 days in the fridge. However, the fluorescence after 4 days was significantly less than the initial fluorescence.

Control                               Initial Test                         After 4 Days

Light-Induced Lysis