SDS-PAGE: Protein Assay

Yesterday (January 31st, 2013), I went to RPI and worked with Eun Ji. But before that, I stopped by the Union Building, got a bit lost, and picked up my ID card (FINALLY!!!! whew...).

When we were heading to the lab, I told Eun Ji that I just did DNA electrophoresis in my bio class. Then, she said that she was just going to do an electrophoresis to confirm her previous data and asked me if I want to do it with her (Of course!!:D)!

Gel electrophoresis is a coomon used technique in biotechnology. It can separate and determine the size of the DNA fragments (cut by restriction enzymes) according to their moving rate. First we load the DNA fragments into a gel well. Then, a positive electric current passes through the other end of the gel. Since DNA fragments are negatively charged, they will slowly migrate to the positive end of the gel at a rate that are proportional to their sizes (the smaller the faster).

However, instead of doing DNA electrophoresis, Eun Ji and I were doing the protein electrophoresis, or SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). Unlike DNA, protein has a very specific 3D shape where all the amino acids are tightly packed. Thus, first of all, we have to make it into a straight chain of polypeptide by adding DTT or other substance, which break the hydrogen bonds between amino acids. Then, we have to add SDS to make the positive-charged protein negative charged because gel electrophoresis works for substance to move from negative end to positive end.

Yet, Eun Ji and I were working on the step before launching electrophoresis - the protein assay, a
simple and accurate procedure for determining concentration of a protein sample. This step is critical because when we are loading the protein into the gel, we want each lane to have the same amount of protein (μg) , not the same volume (μl). Those protein samples are extracted from E. coli. However, while most proteins exist in cytosol, some are in cellar compartments such as inclusion body that are hard to extract proteins. The reason why she wanted to double check her is because in order to get the protein, she has to add SDS to break the inclusion body, which would also affect the targeted protein itself.

Therefore, first of all, Eun Ji got all the materials and the samples she wanted to re-investigate  Also, we want to make sure that we do wear gloves because since SDS affects protein, it can also destroy our skin (It smells really bad, and being exposed too long under it can cause health effects, like I got an headache for just smelling for a few seconds). We divided the 96-well plates into a few sections.

For lane 1,2,3 we added different known concentrations of bovine serum abumin standards as marker (from 0 μg/μl to 5μg/μl). Lane 4, 5, 6 are protein extracted from cytosol, and 7, 8, 9 from inclusion body (which appear blue because of the SDS). Each well in lane 4, 5, 6, 7, 8, 9 were added 10μl of protein samples. Then, we add 200μl of diluted dye reagent to each well, and incubated for about 20 min. The solution turn its color, and we can tell the relative concentration by just comparing the color of the samples to that of the standard. Noted that one can't really distinguish the differences in SDS lanes (7/8/9), so the data analysis wouldn't be reliable.

The next step would be to put the plate into spectrophotometer to measure the absorbance and calculate the concentration. However, because of the shortage of time, I wasn't able to get to this step:( Nonetheless, Eun Ji explained the following steps, converting volume, loading and running gel, and analyzing gel, in which the whole process may take up to 2 days!:O To get the specific target protein even involves more steps!

Later in the day Eun Ji sent me the data including some comments. In general, I did fine on it, but compared to hers, my numbers fluctuate a lot. One thing I would need to work on is the pipette technique, especially when dealing with such tiny amount of substance. Meanwhile, I asked Eun Ji why SDS-PAGE runs vertically where as DNA eletrophoresis runs horizontally. She was kind of surprise because she has never thought about this before!! In the email, she explained that SDS-PAGE is consist of two layers (upper layer (stacking gel) and lower layer (separating or running gel), and vertical direction just makes the process easier. Another reason that I found online is that since the gel runs best in anoxic environment, it's better to have the protein in sealed b/w 2 layers rather than open layer like that in horizontal one.

Anyways, I really learned a lot of lab techniques yesterday, and Eun Ji was always really patient and did a good job explaining things to me. Next week I can finally get into CBIS on my own!!!:D

Intermittent Dosing of Drug Interferes with the Development of Drug-Resistant Melanoma Cells

Last Wednesday (January 23, 2013), I wasn't able to go to my internship because of the Community Service Day. However, I read an article on Medical Daily about that new discovery shows intermittent dosing of anti-cancer drug could interferes with the development of drug-resistant melanoma cells.

Melanoma is the most aggressive type of skin cancer. Researchers have identified mutations in a gene called BRAF that causes normal cells in the body to undergo transformations, accumulate, grow into tumors, and spread. In 2011, the U.S. Food and Drug Administration approved Vemurafenib (Zelboraf) for treating patients in late-stage melanoma with BRAF mutations. Although the tumors initially shrink, the effectiveness does not last long. The cancer cells would become drug-resistant, or "addicted to the drug", by making more of the BRAF protein.

However, researchers are able to use this "addiction" property to combat the cancer. They explain that intermittent dosing of vemurafenib works because cancers not only develop resistance but also dependency on the drug. Thus, when the drug is temporarily removed, the tumors start to shrink. More studies have been done in mice, in which researchers revealed that "mice continuously treated with vemurafenib all died of drug-resistant disease within about 100 days, while those treated with vemurafenib but with regular breaks all lived past 100 days." While no cure has been found, this intermittent dosing treatment could certainly prolong the lives of patients and give them hope. (The link to the article is: http://www.medicaldaily.com/articles/13821/20130111/drug-resistant-melanoma-cells-become-addicted-cancer.htm)

Recently, I become very interested in cancer because some of my family members had suffered from cancers. I think this article is very interesting as it contradicts what we usually thought - diminishing cancer cells by not treating them with drugs. Although it's my first time to really learn about skin cancer, I believe, similar mechanism could also apply to other types of cancer. I really hope to learn more about them and hopefully do some research on them in the future!

After 2 weeks, I am finally be going to my internship this Wednesday (woohoo!) I can't wait to know what I will be doing:)

Bacterial Transformation Lab

Last Wednesday (January 16, 2013) I couldn't go to my internship because of the heavy snow:(

However, luckily, we were doing a bacterial transformation lab in my AP biology class, and it is basically what my internship is doing. Transformation is when bacteria intake a piece of foreign gene and express the new gene. In class, we inserted an engineered plasmid (pGLO)containing both a gene that codes for Green Fluorescent Protein (GFP) from a bioluminescent jellyfish and the gene for antibiotics (ampicillin) resistance into E. coli. The transformed bacteria could grow on LB plate (minimum nutrient) with ampicillin and would glow under UV light if arabinose (a type of sugar) is provided.

The brief procedures are:

1. Label the two micro test tubes "+pGLO" and "-pGLO".
2. Use pipettes to transfer 250μl of transformation solution (CaCl2) into both test tubes. 
3. Use a sterile inoculation loops to pick up a single colony of E. coli and added to the test tubes (one colony in each by using a new loops every time). Then, place the tubes in the ice (It was funny how we just walk out and fill the cup with a pile of snow!).
4. Use a new loop to withdraw a film of plasmid and add it to the "+pGLO" tube but not to the other. Softly mix the solution.
5. Place the tubes in ice for 10 minutes.
6. Meanwhile, label the four agar (nutrient) plates: "+pGLO LB/amp/ara", "+pGLO LB/amp", "-pGLO LB/amp", and "-pGLO LB"
7. After 10 minutes, quickly transfer the tubes into the 42C water bath, a process called "heat shock". Leave the tubes in water bath for 50 seconds, and place them back on ice for 2 min.
8. Use a new sterile pipette for each tube and transfer 100μl to the appropriate plate. 
9. Use a new loop for each plate to spread the suspensions evenly on the surface of the agar. Close the plates and check the result after a few hours.

At (5) and (7) when we put the bacteria in ice, they close their pores and go to dormant mode. When we heat shock them, their pores suddenly open and take in the inserted DNA. Thus, it is very critical to follow the protocol during heat shock steps.

The next day we checked our results!!

There are "colonies" of E.coli on both +pGLO LB/amp/ara and +pGLO LB/amp plates. We know those bacteria are transformed because they are antibiotic resistant. Yet, only bacteria on +pGLO LB/amp/ara can glow under UV light because they were given arabinose. There is bacteria "lawn" in -pGLO LB since the plate contains no ampicillin and any bacteria can live. Plate -pGLO LB/amp was clear because none of the bacteria were transformed, and thus, they are not antibiotic resistant.

This lab demonstrates one of the major techniques we use in biotechnology. It also shows how amazing the universality of genetic codes are!

Lastly, this is my glowing bacteria!!:D

This Wednesday we are having a school service day, so I won't be able to go to my internship. However, I am very excited about what I am going to do next!:)

Maximizing resveratrol production in engineered E. coli.

Last Wednesday (January 9, 2012), I started my first day of the second semester with my internship:)

Since Eun Ji was still on break, I worked with Namita. But because she was busy with writing a paper recently and didn't do much lab work, she went over another paper published by Koffas group with me. The paper is about ways and factors that help to maximize resveratrol production in engineered E.coli., and here's the link to the paper--> http://www.ncbi.nlm.nih.gov/pubmed/21441338

Resveratrol is a natural products that is suspected to be responsible for a decreased risk of heart disease and diabetes. It is usually found in red wine, bushberries, peanuts, cranberries, etc. However, all those plants carry only a extremely tiny amount of resveratrol. Thus, the lab examined ways to optimized the production of the molecule.

Factors examined:

• E. coli. strain
• malonyl-CoA availability
• STS, 4CL 
• gene expression
• cerulemin
• promoter system

First of all, the general metabolic pathway for resveratrol production is:

E.coli is the most common used bacteria because there are more documented information about this bacteria, many strands are non-pathogenic, and they act closer to human cells.

There are 2major enzymes involved: 4CL and STS. The amount of the enzymes affects the efficiency of resveratrol synthesis. The group also searched for a more active STS that would increase production rates and levels. They identified 7 new STS, yet further research still need to be done.

One reason why resveratrol is produced in such low amount is the lack of malonyl-CoA. Malonyl-CoA is an important precursor (added reactant) because it is a key molecule for fatty acid biosynthesis. Thus, the group added cerulemin to slow down the biosynthesis of fatty acid, so there will be extra CoA for the production of resveratrol. The group chose slow down instead of cutting the fatty acid pathway completely because fatty acid is essential for life (cell membrane, etc.), and eliminating it entirely may cause negative effects.

What I think more interesting is the expression construct. An expression construct, or expression vector, is usually a plasmid or virus designed for protein expression in cells (such as production of insulin in bacteria). You can also delete a gene in a cell. First, you would design a particular sequence that code for the protein you want the bacteria to synthesize for you. The ends of the designed sequence (gene 1) must match the ends of the target sequence you want to delete (gene 2).

Then, you add recombinase, a genetic recombination enzyme. Recombinase would then switch the two genes. To test whether the genes are switched, we add antibiotic resistant gene in the designed vector. If the result bacteria grow on ampicillin, then that means bacteria successfully intake the designed gene. Here is a nice video that explain the detailed process, but the first 20 sec is the main idea --> http://www.youtube.com/watch?v=d4PFp43brvI

In the end, the Koffas group maintained to achieve a production of resveratrol of 2.3 g/liter (1.4 g/liter without adding cerulenin), compared to  4 μg/g of dry peanuts and grapes and 2 mg/liter of red wines.

Reading this article is quite hard, but I'm glad that I understand the basic concepts of it. Namita also gave me another article to read in prepare of the experiment we will do next week:)