Difference between revisions of "Team:IngenuityLab Canada/Results"
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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. | 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. | ||
</p> | </p> | ||
| − | <div class="article-img" style="width: 100%; height: auto;">https://static.igem.org/mediawiki/2016/1/1e/T--IngenuityLab_Canada--O2evolution.jpg</div> | + | <div class="article-img" style="width: 100%; height: auto;">https://static.igem.org/mediawiki/2016/1/1e/T--IngenuityLab_Canada--O2evolution.jpg</div> |
| + | <h3>Electron Acceptor Substrate Determination:</h3> | ||
| + | <p> | ||
| + | <div class="article-img" style="width: 100%; height: auto;">https://static.igem.org/mediawiki/2016/5/5e/T--IngenuityLab_Canada--ElectronSubstrates.jpg</div> | ||
| + | <div class="article-cap"><strong>Figure 1. Assorted Substrate Concentrations Effect on Oxygen Evolution of PSII. </strong> 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. </div> | ||
| + | </p> | ||
| + | <h3>DCPIP Summary:</h3> | ||
| + | <p> | ||
| + | <div class="article-img" style="width: 100%; height: auto;">https://static.igem.org/mediawiki/2016/8/80/T--IngenuityLab_Canada--DCPIPfigONE.jpg</div> | ||
| + | <div class="article-cap"><strong>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.</strong> 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. </div> | ||
| + | <div class="article-img" style="width: 100%; height: auto;">https://static.igem.org/mediawiki/2016/b/b3/T--IngenuityLab_Canada--DCPIPfigTwo.jpg</div> | ||
| + | <div class="article-cap"><strong>Figure 2. 2.5ug of Photosystem II assayed with 300uM DCBQ.</strong> 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). </div> | ||
| + | <div class="article-img" style="width: 100%; height: auto;">https://static.igem.org/mediawiki/2016/f/f4/T--IngenuityLab_Canada--DCPIPfigTHREE.jpg</div> | ||
| + | <div class="article-cap"><strong>Figure 3.</strong> 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).</div> | ||
| + | </p> | ||
| + | <h3>Gel Analysis:</h3> | ||
| + | <p> | ||
| + | <div class="article-img" style="width: 100%; height: auto;">https://static.igem.org/mediawiki/2016/d/d0/T--IngenuityLab_Canada--GEL.jpg</div> | ||
| + | <div class="article-cap"><strong>Figure 3.Purified Photosystem II protein subunit analysis.</strong> 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.</div> | ||
| + | </p> | ||
</div> <!-- tab2 --> | </div> <!-- tab2 --> | ||
</div> <!-- wrapper --> | </div> <!-- wrapper --> | ||
{{Team:IngenuityLab_Canada_Footer}} | {{Team:IngenuityLab_Canada_Footer}} | ||
Revision as of 03:14, 20 October 2016
Project 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:
GEL 1
Linearization by Oligos Digestion
GEL 2
DNA Origami Transmission Electron Micrographs:
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.
