Analysis of Glom
1. What is Glom?
The field of molecular biology has advanced greatly in recent years. Now, we are able to understand biological phenomenon by studying molecules. In other words, we can know which DNA or proteins are causing the phenomenon to happen by analyzing the cell. Researchers are now trying to discover the cause of biological phenomenon by studying various kinds of DNA and protein.
Glom is a protein found by Japanese researchers led by Professor Sasaki in 2003. It is a protein that binds to Mitochondrial DNA (mtDNA) inside Physarum polyephalum (slime mold). In general, replication and transcription are suppressed when DNA is condensed. However, research shows that Glom causes intense chromatin condensation in the mitochondrial nucleoid (mtnucleoid) without suppressing DNA functions.
2. The purpose of our experiments
In our project, we are trying to analyze different features of Glom by using GlomI (full length of Glom), GlomII (N-terminal of Glom), and GlomIII (C-terminal of Glom). The experiments that we have conducted are the first step to understand this protein and how each domain works.
We used two basic methods of molecular separation to identify the size of Glom. The objective of experiment one was to determine the number of base pairs of Glom DNA by using agarose gel electrophoresis. In experiment two, we tried to find out the size of the protein by SDS-PAGE. In both experiments, we did not know which sample represented which Glom.
3. Preparation for the experiments
We prepared four types of bacteria (Escherichia coli; XL1-Blue) which had a plasmid (pQE-50; Fig.1) transformed in it. Three of the plasmids each had rGlomI, rGlomII, and rGlomIII inserted in it. There was nothing inserted in the fourth plasmid.
These bacteria were separately cultivated with an antibiotic called ampicillin according to the plasmid inserted. We added ampicillin in order to cultivate the bacteria that we wanted (the bacteria with plasmid have a resistance to ampicillin).
Fig.1 Plasmid pQE-50 [i]
[i] Image taken from http://www1.qiagen.com/literature/vectors_pqe.aspx
4. Experiment one
In order to carry out gel electrophoresis, we needed to attain large amount of Glom DNA. Therefore, we used the Polymerase Chain Reaction (commonly known as PCR).
First, we made a solution which consists from DNA polymerase, DNA primers, and dNTP. We added small amount of bacteria into each solution. These solutions were then put into a thermal cycler that repeats the synthesis cycle. We were able to obtain sufficient amount of DNA fragment that we were looking for.
DNA samples that have been amplified were placed into slots made in an agarose gel. After placing in all of the samples, we ran an electric current through the agarose gel for thirty minutes. Then we dyed the agarose gel with ethidium bromide (EtBr ).
5. The result of experiment one
Fig. 2 shows the agarose gel which has been stained. We applied ultraviolet rays to see where each DNA samples are assembled
From the left, each lane represents marker, rGlomI, rGlomIII, rGlomII, vector only, negative control (DW), and positive control. From this result, we estimated the number of each base pair as
GlomI － About1000bp, GlomII － About580bp
GlomIII － About500bp
Fig.2 Agarose gel stained with EtBr
6. Experiment two
In this experiment, we started by cultivating bacteria. The four bacteria were nurtured in separate test tubes filled with culture medium for one night. On the following morning, we took 1ml from each test tube to different centrifuge tubes and used a centrifugal separator to gather the bacteria(time=0). After that, we added IPTG to the original test tubes. IPTG is an inducer which makes Glom DNA express themselves. We did the same operation to each test tubes both two hours (time=2) and four hours (time=4) after adding IPTG.
SDS-PAGE is an acronym for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Proteins are denatured by sample buffer which is added to each sample. We needed to denature proteins so that they can be separated by gel electrophoresis according to their size. Similar to experiment 1, we placed the samples into each slots made in the polyacrylamide gel. After running an electronic current on the gel for fifty minutes, we dyed the gel with CCB and decolorized it until we were able to observe the protein bands.
7. The result of experiment two
Fig.3 shows the result of SDS-PAGE. From the left, each lane represents marker, vector only (time=0,4), GlomII (time=0,4), GlomIII (time=0,4), and GlomI (time=0,4). By observing adjacent lanes, we were able to know which protein was induced by IPTG.
Fig.3 Polyacrilamide gel stained with CCB
The result shows the approximate molecular mass of each Glom protein.
Glom I － About 46,000 GlomII － About 29,000
Glom III － About 18,000 (not clear)
From these experiments, we were successful in acquiring the size data of each Glom sample.
. For the next step, we are planning to observe whether the chromosome of Escherichia coli condense when Glom expresses by using DAPI to dye the nucleoid. This experiment allows us to know which domain is related to condensation. The goal of our project is to see how Glom plays an important role in condensing mtDNA inside mtnucleoid by using DAPI to dye the nucleiod and putting fluorescent antibodies along the internal membrane which has antiporters.
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