Revision as of 03:38, 20 October 2016

Ingenuity Lab - dNANO

Project Results

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:

T--IngenuityLab_Canada--DNA_Origami_Folding_Reaction.jpg

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 1: 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.

Figure 2: 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

Figure 3:Obtaining linear ssDNA 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.

GEL 2

Figure 4: 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:

Figure 5:

Figure 6:

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.

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.

Electron Acceptor Substrate Determination:

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:

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.

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).

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:

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.

@@ Line 14: / Line 14: @@
 		</p>
 	<h3>Self-folding Reaction:</h3>
 		<div class="article-img" style="width: 100%; max-width: 750px; height: auto;">https://static.igem.org/mediawiki/2016/c/c8/T--IngenuityLab_Canada--DNA_Origami_Folding_Reaction.jpg</div>
+<div class="article-cap"><strong>Figure 1: DNA Origami Reaction </strong> 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 </div>
 		<div class="article-img" style="width: 100%; max-width: 750px; height: auto;">https://static.igem.org/mediawiki/2016/6/65/T--IngenuityLab_Canada--Linearlization_Origami.jpg</div>
-		<h3>GEL 1</h3>
+<div class="article-cap"><strong>Figure 1: Flow Diagram of origami reaction using ssDNA from Lambda Exonuclease digestion of PCR amplified product. </strong> 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.
-		<div class="article-img" style="width: 100%; max-width: 500px; height: auto;">https://static.igem.org/mediawiki/2016/d/d8/T--IngenuityLab_Canada--dna1.jpg</div>
+ </div>
-		<div class="article-img" style="width: 100%; max-width: 500px; height: auto;">https://static.igem.org/mediawiki/2016/d/d2/T--IngenuityLab_Canada--dna2.jpg</div>
+<div class="article-cap"><strong> PCR Amplification and Development of ssDNA </strong> 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.
+ </div>
+		<div class="article-img" style="width: 100%; max-width: 500px; height: auto;">https://static.igem.org/mediawiki/2016/d/d8/T--IngenuityLab_Canada--dna1.jpg</div>
+		<div class="article-img" style="width: 100%; max-width: 500px; height: auto;">https://static.igem.org/mediawiki/2016/d/d2/T--IngenuityLab_Canada--dna2.jpg</div>
+<div class="article-cap"><strong>Figure 2: </strong> 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.
+</div>
 <h3>Linearization by Oligos Digestion</h3>
 <div class="article-img" style="width: 100%; max-width: 750px; height: auto;">https://static.igem.org/mediawiki/2016/8/87/T--IngenuityLab_Canada--dna8.jpg</div>
+<div class="article-cap"><strong>Figure 3:Obtaining linear ssDNA </strong> 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.
+ </div>
 	<h3>GEL 2</h3>
 <div class="article-img" style="width: 100%; max-width: 750px; height: auto;">https://static.igem.org/mediawiki/2016/4/4d/T--IngenuityLab_Canada--dna7.jpg</div>
+<div class="article-cap"><strong>Figure 4: </strong> 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.
+ </div>
 <h3>DNA Origami Transmission Electron Micrographs:</h3>
 <div class="article-img" style="width: 100%; max-width: 750px; height: auto;">https://static.igem.org/mediawiki/2016/9/97/T--IngenuityLab_Canada--dna.jpg</div>
+<div class="article-cap"><strong>Figure 5: </strong> </div>
 <div class="article-img" style="width: 100%; max-width: 750px; height: auto;">https://static.igem.org/mediawiki/2016/e/eb/T--IngenuityLab_Canada--dna6.jpg</div>
+<div class="article-cap"><strong>Figure 6: </strong> </div>
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