Part:BBa_K1499004:Experience

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Applications of BBa_K1499004

Group: 2016 Stanford-Brown iGEM Team

Author: Michael Becich

SDS-PAGE Gel Purification of CBD-Streptavidin Linker--Protein Band Shown in Duplicate at 39 kDa

Summary: The 2016 Stanford-Brown iGEM Team purified this linker protein and used it to create a BioDevice. Used in tandem with a biotinylated fluorophore, this CBD/Streptavidin fusion protein served as a linker between cellulose paper and the fluorophore-quencher biosensor described here: http://2016.igem.org/Team:Stanford-Brown/SB16_BioSensor_FQsensor.

Once we were able to achieve a working biosensor prototype, our next step was to utilize this in a scenario applicable to embedding in our bioballoon. We decided that cellulose sheets would serve as a satisfactory surface for proof of concept, knowing that later down the road, we could use different binding domains for latex, elastin, collagen, or p-aramid fibers. Conveniently the 2014 Stanford-Brown-Spelman iGEM team had created a Cellulose Cross Linker BioBrick BBa_K1499004 that needed further characterization. We filled this need by purifying the protein (validating the presence of its HisTag), and binding our fluorophore sensor to the linker protein (with quencher).

Depiction of our ATP Sensor Biodevice

We then distributed this incubated concoction to wax-coated cellulose filter paper to measure the binding activity to the paper over a week. Initially the mixture was applied to the paper and per recommendation 2-3 days is necessary for the cellulose binding domain to take effect. After this initial binding period, 5 x 1mL of milliQ water (with 1mM ATP) were washed over each 9-well sample each day for a week. The positive control had the FQ system, but no linker. The negative control had no FQ either. Fluorescence was also quantified on the Typhoon scanner for characterization purposes. In order to confirm that our biodevice was working, we measured the fluorescent activity on a cellulose sheet over the course of a week.

The depicted timelapse gives qualitative proof that both the FQ sensor and Cellulose Cross-linker are working in tandem, as evidenced by the drastic difference in fluorescence between the experiment and controls, amplified from day 1 to day 7.

Fluorescent images were analyzed with Typhoon Scanner (Absorption=495nm, Emission=525nm) and processed quantitatively with Python.

Each day 5x 1mL 10mM ATP was pipetted onto each 9-well grid to simultaneosly wash off unattached sensors and trigger attached sensors. We understand that photobleaching and heterogeneous mixing across the wells are uncontrolled for, but this pilot study gives way for proof of concept embedded abiotic nucleic-acid based sensors.

User Reviews

UNIQac92b656cfbff79a-partinfo-00000000-QINU

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mbecich

The part worked as specified for our needs! It should be noted that an inducible promoter, HisTag, and double terminator are all included in this part to facilitate expression and purification. This was confirmed to us by the part creator and former Stanford-Brown iGEMer herself, Alaina Shumate.

Group: 2016 INSA-Lyon iGEM Team

Author: Mathieu Borel

Summary: The 2016 INSA-Lyon iGEM Team purified and characterized this part. The team showed it was possible to purify this part using affinity chromatography on a cellulose column. With a biotinylated and Fluorescent labelled DNA oligo the team also showed it was able to bind at the same time cellulose and biotin. You can see our proof of concept page for further details on how we used this part in our system: http://2016.igem.org/Team:INSA-Lyon/Proof

We also modeled the chimeric protein, you can download the PDB file here

INSA Lyon 2016 Experiments on this part

Characterization

1. Purification Using Cellulose Affinity

The BBa_K1934020 part conceived by the 2016 INSA-Lyon team and synthesized by IDT was cloned into pSB1C3 and transformed into the E. coli NM522 strain. One recombinant clone was grown overnight in LB at 24°C, with IPTG 1 mmol/L^-1 and glucose 5 mmol/L^-1. Cells were harvested and resuspended in 1 mL lysis buffer (50 mmol/L^-1 Tris, 300 mmol/L^-1 NaCl, 10% glycerol). Then the mix was sonicated 5 times 30 seconds on ice at moderate power. The lysate was centrifuged at 14,000 g for 10 min. The supernatant was treated as follow:

Wash microcrystaline Cellulose five times in water. Then equilibrate in washing buffer (ammonium sulfate 1M). Pack the cellulose (10x10mm) in small chromatography columns (we used syringes barrels).
Gently pour the lysate supernatant on the column. Once the liquid starts flowing through evenly, measure the OD₂₈₀ of the different fractions. Continue pouring washing buffer until the OD₂₈₀ stabilizes around zero.
Change the washing buffer to water. OD₂₈₀ shortly rises. Keep the fractions with the highest OD₂₈₀ . They should contain the protein.
Analyse collected fractions on an SDS-PAGE. Optionally, proteins may be concentrated using ultrafiltration.

**Figure 1. Purification of the chimeric Streptavidin-CBD protein on a cellulose column** This elution graph shows a first peak, present for both the control and our expression culture. This first peak corresponds to unbound proteins. In the presence of water, only one peak was observed: it’s the elution peak of our protein.

2. BBa_K1934020 encodes a protein able to bind both biotin and cellulose

Affinity of the streptavidin-CBD encoded by BBa_K1934020 to cellulose was compared to the one of commercial streptavidin. A molecule of fluorescein was grafted at the 5’ end of a DNA oligo carrying a molecule of biotin at its 3’ end. This DNA oligo constitutes the reporter system. Such modified oligo was mixed either with the engineered streptavidin-CBD or with commercial streptavidin. The resulting mix was incubated with microcrystalline cellulose in presence of PBS for 1 hour. The cellulose was then washed twice with fresh PBS and fluorescence was measured. Every experiment was done in triplicate.
**Figure 2. The Streptavidin-CBD is able to bind biotin and cellulose.** Mixed raw cellulose with our report system shows no fluorescence (first bar). The measured fluorescence indicates that commercial streptavidin was able to bind our reporter system and sticks at a low extent to cellulose. We concluded that this results from none-specific adsorption. For the streptavidin-CBD part (BBa_K1934020), a high fluorescent signal was recorded.
**This experiment shows that this streptavidin-CBD protein is able to bind efficiently biotin and cellulose at the same time.** The same experiment was done for the BBa_K1934030: part displaying a different cellulose binding domain, namely CBD-CipA. The binding efficiency of streptavidin-CBDs tends to be slightly lower compared to streptavidin-CipA (x1.1) but was not statistically demonstrated.
3. Streptavidin-CBDs modeling

We made a homology modeling as a confirmation of the working protein folding. The domains don't seem well defined because of the hindered, but we still conserve the secondary structure of the protein. The use of a linker may be appropriate to allow a better efficiency.