Team:IngenuityLab Canada/Results

Ingenuity Lab - dNANO

 

DNA Origami Results
Photosystem II Results

DNA Origami Results

Obtaining scaffold DNA:

The first test we did was to optimize our PCR protocol to achieve DNA product that is only of the segment that is utilized in our structure. This allows us to omit the DNA segment from the M13mp18 DNA that is not being utilized. You can see the gel 1 (below left) shows the not optimized PCR while the Gel 2 (below right) shows the optimized PCR product. A single thin and bright band at approximately 7000 BP shows us that the sample analyzed on Gel 2 is almost fully optimized, as oppose to the not optimized version.

Self-folding Reaction:

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Figure 1: DNA Origami Reaction. The DNA Origami reaction is performed using 1:10:1 ratio for M13mp18 linear ssDNA concentration, individual staples concentration and capture strands, respectively. The concentration and reaction parameters were adapted from Dr Seidel’s group. The mixture of purified linear ssDNA M13mp18 is mixed with staples and Folding Buffer in a single pot and heated to +80OC and cooled slowly over 15 hours using non-linear temperature ramp. Using Agarose Gel Electrophoresis, we analyzed the folding structure and TEM imaging we obtained multiple structures that fit the size of our model design. These structures were aligning head to tail when viewed under the TEM (Figure).



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Figure 2: Flow Diagram of origami reaction using ssDNA from Lambda Exonuclease digestion of PCR amplified product. The above flow diagram is a schematic of the steps for the development of a hollow prism origami structure. The Process uses RFII plasmid DNA of m13mp18 bacteriophage as a template. This template is then reacted with two primers flanking the desired origami template sequence with the reverse primer being phosphorylated. Using Phusion Polymerase, a 7kp PCR product is formed containing the template strand. The following digestion of the phosphorylated strand is conducted at with lambda exonuclease at 37 degrees for 1 hour. The ssDNA product is then purified either by spin column or phenol:chloroform extraction. The purified product is then mixed at a concentration of 1:10 template strand to origami staples and is cooled for 15 hours on a nonlinear gradient in a thermocycler.
PCR Amplification and Development of ssDNA Our project initially began using the Replicative Form plasmid of M13mp18 phage. PCR amplification was conducted on the plasmid to form a ~7kb origami template sequence. Figure 2 shows the the expected band size of the PCR products. Other bands are also seen at approximately 3500 bp and 1500 bp. The primers when designed were not reviewed for nonspecific binding to the RF plasmid before being ordered.
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Figure 3: Digestion of the PCR product was conducted multiple times but did not yield high enough DNA concentration to be able to be used for downstream application in the origami reaction. Gel electrophoresis was used to confirm the digestion of the PCR product by lambda exonuclease. Loaded samples were not able to be visualized on the gel (data not shown) and proper digestion could not be confirmed. After multiple trials, and attempts to purify the lambda exonuclease digest, the method to generate ssdna was refined and Oglionucleotide digestion with Bsrb1 was determined to be the best course of action.

Linearization by Oligos Digestion

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Figure 4: After multiple attempts to obtain linear M13mp18 ssDNA using PCR and subsequently treating it with Lamda Exonuclease, we decided use an alternative method that Paul Rothemund utilizes when performing DNA Origami. Circular M13mp18 ssDNA from New England Biolabs and a complimentary oligonucleotide that will bind to M13mp18 ssDNA from IDT. The location of binding in the circular M13mp18 ssDNA strand is not utilized for our designed origami structure and forms a specific restriction digestion site within the binding region, BsrBI. Analysis by nano-drop shows much higher concentration retrieval. Phenol:Chloroform extraction yielded greater than 60% recovery after digestion over multiple attempts. Concentrations were consistently over the 10 mM required for 50 uL origami reactions, data not shown. compared to the method which required PCR Followed by Lamda Exonuclease digestion. Using the linearized M13mp18 ssDNA we performed the Origami reaction.


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Figure 5: During attempts to linearize using lambda exonuclease, pure circular ssDNA of the M13mp18 bacteriophage was used to directly in the origami reaction. No proper structures were expected to form however, better than expected results occurred (Figure 5 d-f). Structure formation was confirmed by gel electrophoresis in TBE buffer supplemented with 11mM of MgCl2. Figure 4 shows bands in lanes 4, 5, and 6 which move slower than the circular ssDNA control in lane 1. Respectively dsDNA PCR product was loaded in lane 2 to confirm the faster migration of the expected origami structures due to increased compactness of the DNA. The loaded staple concentration lanes 4 and 5 are also significiantly reduced when compared to the staple concentration control in Lane 3.

DNA Origami Transmission Electron Micrographs:

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Figure 6 Comparison of the folding reactions from both the BsrbI digestion (6b and 6c) and the circular ssDNA (6e and 6f) with a heat control in which ni staples were added (6a). Compared to the unreacted ssDNA (6d) macrostructure formation is seen. In reaction samples long strands of organised structures are seen distinctly at micrometer scales. Figure 6d shows no organization in structure overall and similar to the heat control in Figure 6a.
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Figure 7 The formation of the expected double wall prism of the origami structure compared to the literature sources. Figure 7a is unreacted ssDNA showing no organized structeres but a mesh with no matter. Figure 7b shows a magnified view of linear ssDNA showing the unexpected binding that is forming the DNA origami macrostructures. Figure 7c shows the expected single units formed from circular DNA and were designed using caDNAno . Structurally they can be seen to have a double-sided wall surrounding a hollow cavity. Length and width also match expected parameters of the designed structure. Similarly figures 7d and 7e also show the single unit structures forming, as well as the macrostructures. The macrostructures in figures 7d and 7e can be seen to contain a repeating series of bulging areas that correlate to the length of the individual origami units


Photosystem II Results

Growth of the HT3 cells:

The cells were grown in 10 L batches in order harvest largest amount of protein. Below shows the data of growth measured using absorbance at 730 nm. Each day, the data was collected until it reached approximately 0.8 and not higher than 1. Below the graph indicates the exponential growth rate of the HT3 cell grown in BG11 media.

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The graph indicates the exponential growth of the HT3 cells grown in 10L culture of BG11 media.

Purification of Photosystem II:

Throughout the purification, we saved sample from each step and analyzed its oxygen evolving ability in comparison to its chlorophyll a concentration. This was done to ensure that at each step we are following and optimizing protocol in order to achieve the most active photosystem II protein. Below the graph shows the oxygen evolution rate in comparison to the sample’s chlorophyll a content.

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Electron Acceptor Substrate Determination:

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Figure 1. Assorted Substrate Concentrations Effect on Oxygen Evolution of PSII. 150μM, 300μM and 500μM concentrations of substrates DCBQ (2,6-dichloro-1,4-benzoquinone), CoQ1, CoQ4, CoQ10, Menaquinone and Decylubiquinone were used to test their effect on the rate of oxygen evolution of PSII (%). The rate of oxygen evolution of PSII (umol/hr/mg Chlorophyll a) was normalized to the activity of 300uM DCBQ and converted to percent activity. 300 uM DCBQ revealed to have the highest rate of oxygen evolution, thus it can be used to aid the conduction of electrons from PSII's quinone channel as well as used for proteoliposomes oxygen evolving activity.

DCPIP Summary:

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Figure 1. The electron reduction activity was measured using a redox dye, 2,6 Dichlorophenolindophenol (DCPIP) which can be used to determine rate of photosynthesis by measuring absorbance at 600nm. A standard curve for DCPIP was created in buffer (5mM MES, 50mM KC, 2mM MgCl2, 2mM CaCl2 pH 6.52). The equation, y=158.16x - 4.1787, from the standard curve was used to determine the change in concentration for DCPIP when tested with electron acceptor DCBQ or DQ and Photosystem II protein. As PSII is excited with light, DCPIP replaces the role of NADP+ and accepts an electron from the break down of water. The DCPIP changes from blue to colorless as it is reduced.
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Figure 2. 2.5ug of Photosystem II assayed with 300uM DCBQ. The light was turned on at 0 seconds and the changes in the O2 was detected at 30 second interval for 600 seconds. The Max Rate of Change for 30 muM DCPIP 113.6049 +/- 3861.6103 (umol O2 per hr per mg Chla) and Max Rate of Change for 40 muM DCPIP 194.5256+/- 22.2701 (umol O2 per hr per mg Chla).
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Figure 3. Oxygen rate of proteoliposomes were measure using 30uM DCPIP, 50uM DQ and 25ul of proteoliposomes. Max Rate of Change Rsat PSII 96.9615 +/- 35.3789 (umol per hr per mg Chla) Max Rate of Change Rsol PSII 165.3464+/- 193.0685 (umol per hr per mg Chla).

Gel Analysis:

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Figure 3.Purified Photosystem II protein subunit analysis. Lane 2, 5, 8 is the standard protein ladder, and lane 3, 6, 9 are Photosystem protein loaded with 100ug, 50ug and 25ug of the protein with 8M urea and 5X loading buffer. As shown, all the subunits of PSII are present in the lanes. The are present approximately The molecular weight of PSII protein D1 and D2 subunits are 38.270kDa and 39.390kDa which from lane 3 shows the markers at 36.986kDa and 40.277kDa.

Photosystem II Summary:

Table 1: PSII Purification data summary Chlorophyll A (ChlA) content was determine by adding 10uL of each sample into 80%(v/v) acetone followed by sonication for 3 minutes, centrifuged for 3 minute. The supernatant was removed and the OD663 and OD645 were measured using a Beckman Coulter DU720. If necessary, the pelleted material was subjected to the chlorophyll extraction process 2-3 times. Chlorophyll A content was calculated using equation 1.

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Figure 1: Affinity Chromatogram Normalized to maximum absorbance. Isolated Thylakoid membranes were resuspended to 890ug/mL chlA in Thylakoid Buffer. The membranes were solubilized by drop wise addition of 20% (w/v) DDM to a final concentration of 0.8% (w/v) and incubated at 4°C for 20 minutes. Non-solubilized material was removed by centrifugation using a multiple speed increase from 100rcf x 1 min to 22,100rcf x 10 minutes using a Beckman Coulter Type 45-Ti Rotor All chromatography steps were performed using a GE AKTA Pure 25. Sample was loaded on to a GE XK 20/10 column packed with GE NI2+ Sepharose High performance media pre-equilibrated with binding buffer at a linear flow rate of 38 cm/h (~4mL/min). Column washing was continued with binding buffer at 38 cm/h until Abs280 fell below 90mAU. PSII was eluted using 70mM L-Histidine in up-flow operation at 22.6 cm/h (2mL/min) and 2mL fractions were collected. Fractions 15-22 were pooled and concentrated using a 100kd MWCO Amicron concentrator. Removal of L-histidine was completed by diluting the concentrated sample 10 fold with binding buffer and concentrated. This was repeated 3 times.

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Figure 2: Size Exclusion Data and Fraction Activity Binding Buffer was used as elution buffer and elution was performed at 100ul/min. 100uL of sample was injected using a 100uL loop, 300uL Fractions were collected and Oxygen Evolution activity was performed 3 times for each fraction using 50 mM MES-NaOH, pH 6.5, 10 mM NaCl, 5 mM MgCl2, 20 mM CaCl2 and 0.04% DDM. Ferricyanide was added as electron acceptor at 2mM. The reaction took place at 28°C and was initiated by illumination at 2700 umol/min/m2. The results are the average of three independent tests. The two main peaks both showed activity which indicates the first is PSII dimer and the second is PSII monomer. The calculated molecular weights are in good agreement with PSII bound with DDM micelles.

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Figure 3: Oxygen Evolution Assay Results of 17MAY2016 Purification: 15ug of ChlA in 1.5mL Assay Volume. Assay was performed using 50 mM MES-NaOH, pH 6.5, 10 mM NaCl, 5 mM MgCl2, 20 mM CaCl2 and 0.04% DDM. Ferricyanide was added as electron acceptor at 2mM. The reaction took place at 28°C and was initiated by illumination at 2700 umol/min/m2. The results are the average of three independent tests. The rate of Oxygen Evolution was calculated using a 60s time step.

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Figure 4: Oxygen Evolution Assay Assay conditions are the same as previous figure. However, Ferricyanide concentration was 1mM and 2,6-dichloro-p-benzoquinone was added to a final concentration of 500uM.

Proteoliposomes Summary:

FlexStation 3 Multi-Mode Microplate Reader was used to run the assays on Photosystem II (PSII) proteoliposomes. The test is to monitor the internal pH of the proteoliposomes with Pyranine pH probe upon exposing it to light. Light causes the PSII protein to split 2 water molecules into 4H+, 4e- and 2 oxygen molecules. Based on internal acidification of alkalization, the orientation of PSII can be determined. If the quinone channel is oriented right side up, the expected result is to see the split of water inside the proteoliposomes thus internal acidification.

After adding 175ul of assay and 25ul of proteoliposome sample, an initial reading is taken at λem = 410 nm and λexc= 460 nm. The reading ratio of 460/404 is used to determine the pH value for each sample. In this experiment, a ‘control’ sample that does not contain PSII was used to confirm that the proton activity is only due to the protein integrated in the liposome. In addition, the ‘control’ sample contained 3mM and 7mM CHAPS detergent labelled Rsat (lipid to detergent ratio~ 17.4) and Rsol (lipid to detergent ratio~ 7.5) respectively.

Other controls in the experiment included Valinomycin, an ionophore that balances K+ ions down electrochemical gradient; Ferricyanide (FeCY), an electron acceptor impermeable to plasma membranes are in the assay outside the liposome; p-xylene-bis-pyridinium bromide (DPX) quenches the non-permeant pyranine pH indicator dye reconstituted in the liposome along with PSII protein, which is used to measure florescence intensity with proton activity. 2,6-dichloro-p-benzoquinone (DCBQ) and decylubiquinone (DQ) were the different electron acceptors tested in the assays; DCBQ degrades after long exposure to light, whereas DQ is stable for much longer period of time.

Adding Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) in the assay mixture is uncouple the liposome membrane, break down electrochemical gradient, and balance pH between assay and liposome. As a result, the pH of the sample increases. In this experiment, upon addition of CCCP after the 1020 seconds mark, the expected result is to see the pH going from an acidic state back to a state of equilibrium.

Proteoliposomes were tested in each of the assays made with 5mM MES, 50mM KCl, 2mM MgCl2, 2mM CaCl2 pH 6.52. Final Assay mixture concentrations in 200ul volume. A)300µM DCBQ, 0.2µM Valinomycin, 2mM FeCY, 20mM DPX; B)300µM DCBQ, 0.2µM Valinomycin, 2mM FeCY, 20mM DPX, 1.5µM CCCP; C)50 µM DQ, 0.2µM Valinomycin, 2mM FeCY, 20mM DPX. D)50µM DQ, 0.2µM Valinomycin, 2mM FeCY, 20mM DPX, 1.5µM CCCP

175ul of stock assay and 25ul of liposome= total 200ul volume contained the final assay mixture concentration. Total of three trials were completed for each of the assay mixture. PSII protein from August 23, 2016 was used with chlorophyll concentration at 0.629mg/ml and PSII concentration at 6.29mg/ml

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Figure 1. PSII E.coli Total Lipid Extract Liposome Reconstitution with Pyranine Screening and DCBQ.

The initial pH of the buffer was at 6.52; the 4 samples (Control Rsat, Control Rsol, CHAPS Rsat, CHAPS Rsol) which initially had the 1.5uM CCCP in the assay had pH relatively close to the 6.52 assay buffer value. In other words, the CCCP uncouples liposome that caused the proton gradient to break thus equalizing the pH inside the liposome and outside in the assay. Comparing the group with CCCP to the group without, the data suggests that the CCCP was effective in uncoupling the liposome membrane to some extent. For CHAPS Rsat and Rsol, the delta pH for group with CCCP is higher than without. It is necessary to consider that only 1.5uM CCCP was in the assay, which may not have been enough to cause complete destruction of the proton gradient. Thus, with the CCCP the movement of proton in the assay may be faster. The CHAPS Rsat and Rsol without any CCCP suggests the PSII is incorporated in the liposome as the pH gradually increases upon illumination; After addition of CCCP to CHAPS Rsat and Rsol at 1020seconds~ mark, although initially the pH increases, it stabilizes after the 5 minutes’ incubation supporting the idea that PSII was causing the alkalizing of the liposome as it is oriented upside down.

All the liposomes alkalize upon illumination- water is split to proton outside of the liposome and into the assay; the gradual increase of pH for all the groups excluding Control Rsat/Rsol with CCCP suggests that there is no leakage in the liposome. For all control group, the pH gradually increases by small units (0.03~ units) each time even after the addition of CCCP at 1020 seconds~ time mark. Comparing the control Rsat/Rsol to CHAPS Rsat/Rsol shows that having the PSII small changes in pH with illumination. However, once CCCP is added, the proton gradient causes 0.4 units of pH changes. Overall, using DCBQ as the electro acceptor in the assay suggested that PSII is functionally incorporated in liposomes CHAPS Rsat and Rsol.

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Figure 1. PSII E.coli Total Lipid Extract Liposome Reconstitution with Pyranine Screening and DQ.

With DQ as the electron acceptor, the initial pH of all is resistant to change with the light illumination incubation. However, with longer periods of time in light incubation, the pH for Rsat and Rsol without CCCP increases gradually suggesting the PSII protons are found out of the liposome and is alkalizing the inner core. After CCCP is added at 1020seconds mark, the pH stabilizes.

Although the pH of all the samples increase substantially after the 500 seconds mark, only the liposomes with the PSII have a high delta pH increase. This could potentially be because the PSII is slowly being activated by the light illumination. Furthermore, adding CCCP causes the CHAPS Rsat and Rsol to slow down their proton activity as it reaches a plateau of very little change at the end of the experiment period. An important aspect to note about DQ is that it may not be a fast electron acceptor and is easily affected by uncoupling of proton gradient as the pH does not change much after adding CCCP. CHAPS Rsat with CCCP had the highest delta pH and the alkalizing begins shortly after 300 seconds of incubation in light.

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