The End of Summer

Last week, I finished my work at Princeton. Although it is not the ending of our project, the progress we made this summer is really important for the whole project.

The main part of our improvement includes two parts: thylakoid extraction and photosynthesis efficiency testing.

For the first part, the main procedures include: 1. using a lab blender to blend the mixture of alginate leaf pieces with grinding buffer; 2. putting the solution into the centrifuge for the pellet; 3. putting the pellet into the washing buffer and do the resuspension; 4. go through few more times of centrifuge to get the concentrated thylakoid.

For the testing, the main procedures include: 1. adding 0.10 mL of the suspension to 10 mL of 80% acetone in a test tube; 2. inverting the solution several times and then filtered through a Whatman filter paper into a large cuvette using a 50 mL glass funnel; 3. measuring the absorbance of the green solution using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation. 

We tried multiple methods to see which works the best. There was failure sometimes, but we got through it and kept on moving forward. This project includes a lot of brainstorming and testing. Different from my last project, I have experienced more about the beginning part of a science project, including proposal writing, idea design and other preparation.

Since the project is still in progress, I will go back to Princeton to continue my lab experience during the school year. It is the consistency that makes a difference. 

Week 8 - 10

Hello, this is Jacky Jiang from McAlpine group in Princeton. It has been a long time from last post. In the past three weeks, we keep on testing the efficiency of our methods to make thylakoid and the concentration of our chlorophyll.

To get better concentration, I need to come up with different methods of producing the thylakoid. As I have tried the classic procedures, the basic steps would be similar. First of all, I need to use a lab blender to blend the mixture of alginate leaf pieces with grinding buffer. After we get the solution, we will put it into the centrifuge for the pellet. The pellet we get need to go through another step, which is called resuspension. In this step, we put the pellet into the washing buffer and do the resuspension procedure. Then, we need to go through few more times of centrifuge to get the concentrated thylakoid. To make modification, I tried different kinds of centrifuge rate, which made the composition of the pellet different. This change could be very critical. Since the nuclei and other fragments of plant cells have different density, the centrifuge rate determines which component would be at the bottom of the pellet. What’s more, I also change the grinding buffer I used for blending the mixture. The different concentration of tricine would make the grinding level different, so that the size of membrane fragments would be different, too.

After I tried different methods, the results comes out that the classic steps with appropriate concentration of grinding buffer and high centrifuge rate worked the best. I also tried to change the sequence of centrifuging and buffer mixing, which didn’t turn out well in the end.

 To determine if our thylakoid is efficient enough, we still need to determine it by the concentration of chlorophyll. The experiment methods are the same as we did last time. The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to 10 mL of 80% acetone in a test tube. This solution is mixed by inverting several times and then filtered through a Whatman filter paper into a large cuvette using a 50 mL glass funnel. The absorbance of the green solution is measured at 663 nm and at 645 nm using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation.  

Our thylakoid concentration has improved a lot after we modify the methods. In the rest of the summer, we will move on to the electrical part of our project. 

Week 5 to 7

Hi, this is Jacky and I am working in McAlpine Lab at Princeton. 

These three weeks have been very busy. Our projects have been moving to the first critical point, which is about measuring the quality of the thylakoids we make.

                The first test we perform is chlorophyll concentration measurement. The chlorophyll concentration in the thylakoid suspension is determined by adding 0.10 mL of the suspension to acetone in a test tube. This solution is mixed by inverting several times and then filtered through a Whatman filter paper into a large cuvette using a glass funnel. The absorbance of the green solution is measured at 663 nm and at 645 nm using 80% acetone to zero the spectrophotometer. The concentration of chlorophyll in the original sample is calculated using the relative equation. Once the chlorophyll concentration is determined, the total chlorophyll yield should be determined by multiply the chlorophyll concentration in mg /mL times the volume (mL). Once the chlorophyll concentration and the total chlorophyll yield is known, the chlorophyll concentration should be adjusted by adding the appropriate amount of Washing Buffer or by centrifuging again the thylakoids and resuspending in the appropriate amount of Washing Buffer.

                By determining the concentration of chlorophyll we make, we can decide if the procedures we use is appropriate since there are a lot of different methods that we are able to choose from literature. It is always important in science research to try as much methods as possible.

                                          

                What’s more, we need to test the efficiency of the thylakoids we extract. In this case, we need to use a chemical method called Hill Reaction. As we learned in Biology class, during light reaction photosynthesis, electrons will end up in NADPH. In Hill reaction, we need to separate thylakoid with stroma so that NADP won’t be available. Instead, we will put DCPIP, a blue oxidant, into the solution. DCPIP is blue in its oxidized form, and becomes colorless when it is reduced during the Hill reaction. Thus, the rate at which electron transport occurs in the Hill reaction can be measured spectrophotometrically (at 620 nm) by following the change in absorbance of DCPIP as it accepts electrons from the electron transport chain. To perform the Hill reaction, a sample of a chloroplast suspension will be mixed with the Hill reaction buffer (containing DCPIP) and exposed to light for a series of 30 second intervals. After each exposure period, the absorbance of the DCPIP will be measured. The absorbance values can then be plotted versus time to determine the rate of DCPIP reduction as a measure of PET.

It turns out that the thylakoids we make are pretty efficient. We will keep on testing and trying for next couple days and shift our focus to other aspects. 

Week 3-4 in Princeton

My name is Jacky Ziwen Jiang and I am working in McAlping lab at Princeton for this summer. It has been four weeks and our research has been going pretty well.

As the first two weeks were mainly about planning and chemicals ordering, we turned in to the actual working mode the past two weeks.

The main material we need to prepare is thylakoid, the main factory where photosynthesis takes place. To get thylakoid, the method is not that simple as just cut leaves into small pieces. First of all, we need to choose the right plant leaves for the thylakoid extraction. After reading certain amount of articles, spinach leaves has the relative high concentration of chloroplasts in their leaves, which we will choose for our experiments. After we bought the spinach, there are two more buffers we need to make for the thylakoid extraction. One is grinding buffer and the other one is washing buffer.

The whole process is filled with multiple complex procedures. First of all, the precise measurement of different chemicals we need takes consistent attention without vacillation. After that, I need to use a lab blender to blend the mixture of alginate leaf pieces with grinding buffer. After we get the solution, we will put it into the centrifuge for the pellet. During this process, I also learn new knowledge of the unit conversion between rpm and g, which show the rotation strength. The pellet we get need to go through another step, which is called resuspension. In this step, we put the pellet into the washing buffer and do the resuspension procedure. Then, we need to go through few more times of centrifuge to get the concentrated thylakoid.

The solution looks clearly green and fresh. These two weeks’ experiments give me a great learning opportunity of thylakoid, which gives me a further understanding of the knowledge I have learned in class. This is a great example of practice the knowledge I learned in actual experiments.  

New Project about plant

As we have finished our 3D printed bionic ear, our group is starting a new project. We are basically preparing for the new one, which will be about plant and energy harvesting.

In this project, I need have more reading since I need to contribute a lot to the experiments design and performing, which includes the material choosing and methods design. As I am pretty familiar with the knowledge of plant photosynthesis (thanks to my great AP Biology class), I will be more specifically in charge of the mechanics part of the leaf, while I will learn more about the electronic part.

What’s more, instead of alginate and bovine cells, we will use totally different material for this time, which means we need to design new setting for our lab. These days, through the information we have, we are deciding the material and other electrical components of the project, while the material needs to be reconsidered. We are also preparing for the interview from MIT’s Technology Review, which records the whole process of our 3D printed bionic ear. So a new fresh ear is coming.

One of the most exciting things taking place these past two weeks is the presentation of the new project in our group meeting. This is the presentation that I have longest talk about our idea, in which I explain how our research would go. In the presentation, I first mentioned the necessity of plant energy harvesting and the general information about photosynthesis including photosystem and electron transaction way. Later, I talked about the idea of combining nanotechnology and plant biology, and the specific methods that we were going to follow. The presentation went well.

In the next week, we will start the new project and I cannot wait for the experiments with plant.