User:Saja A. Fakhraldeen
Background
Name: Saja Fakhraldeen
Course: 20
Year of Graduation: 2009
Email: sajafd AT mit DOT edu
- Have you taken
7.05/5.07 (Biochemistry) - No, took equivalent classes at Harvard
7.06 (Cell Biology) - No
7.02 (General Biology Lab) - No
5.310 (General Chemistry Lab) - No
Do you have any experience culturing cells (mammalian, yeast or microbial)? Yes
Do you have any experience in molecular biology (electrophoresis, PCR, etc)? Yes
- Please briefly describe any previous laboratory experience
I have been a UROP at the Engelward Lab (BE department) for the past year. The research I have been working on involves studying the effect of genetics on the rate and frequency of DNA repair. Specifically, the effect of inactivating p53 (a tumor suppressor gene) on the rate and frequency of spontaneous and exogenously-induced homologous recombination.
Genome Engineering Module
- M13 Genome Modification Ideas
| Gene | Function of Corresponding Protein | Re-engineering ideas |
|---|---|---|
| g1 | Assembly | Knockout the gene and individually/simultaneously knockout g4 and g11 and observe whether the virus can still successfully assemble its progeny particles through utilization of the bacterial assembly proteins |
| g2 | Replication of DNA + strand | Modify it such that its protein does not nick the dsDNA without a defined stimulus (when certain conditions within the host cell are satisfied) |
| g3 | Phage tail protein (5 copies) | Modify it such that its protein can be more/less selective of its potential bacterial host (criteria for selectivity can depend on the properties of the host's membrane, the environment surrounding the host, etc...) |
| g4 | Assembly | See idea for g1. Another potentially interesting observation to make using the same knockouts would be the effect that those knockouts have on the growth of the infected bacterial host. |
| g5 | Binds ssDNA | Somehow increase its protein's affinity to M13's ssDNA genome so that it will not have to compete with dsDNA formation |
| g6 | Phage tail protein (5 copies) | Delete the gene and observe whether infection and release can still take place with just the unmodified p3 |
| g7 | Phage head protein (5 copies) | This gene can be useful for adding fluorescent tags (GFP,YFP,etc...) that will allow for real-time observation through fluorescence detection (similar to the video seen in class) |
| g8 | Phage coat protein (2700 copies - can vary with varying length of viral genome) | |
| g9 | Phage head protein (5 copies) | Same idea as g7 - another possible use for fluorescent tagging is relatively easy quantification of the viral progeny which can be useful for studying the effect of different factors on the ability of the phage to successfully complete its life cycle |
| g10 | DNA replication | Modify it such that the promoter is stronger and the gene is transcribed more frequently. That could lead to an accumulation of more dsDNA and it would be interesting to see if that would effect the transcription of g2 and g5 (up-regulation/down-regulation) |
| g11 | Assembly | See ideas for g1 and g4 |
- M13 Relatives
M13 belongs to the filamentous family of bacteriophages. The most extensively studied fF phages (filamentous phages which require the F pilli on the bacterial host for infection) are M13, f1, and fd. One of the differences between these three phages is the distribution of restriction sites throughout their genomes.
- BBa_M1307 as a standard biological part
The part BBa_M1307 in the Registry for Standard Biological Parts is simply a modified version of the M13K07 genome (extra sequence for Kanamycin resistance and a bacterial origin of replication) and thus it does belong in the Registry. This modified version makes it easier for scientists to study/observe the behavior of the phage in the presence of bacteria. It is also useful for scientists who want to engineer modified versions of the phage.
Research Proposal Idea
We hope to test the relative efficacy of various gene silencing techniques.
Currently, one existing technique for gene silencing is to completely eradicate a functional form of the protein translated from the gene (we will refer to this as gene deletion). Another technique is to create mutations in the gene which may lead to variations in the protein, potentially rendering the protein insufficient for its previously intended purpose (we will refer to this as point mutation). The newest gene silencing technique that was discovered is siRNA (we will refer to this as siRNA).
What we wish to test is whether using siRNA is as efficient for gene silencing purposes as gene deletion or point mutation.
We will pick at least 3 different genes (preferably genes that have validated siRNA sequences and genes for which there exist knockout mice/strains to use for the gene deletion category), design at least 3 different siRNA sequences targeting various parts of each of the genes (promoter, ORF, etc...), and, using well-established techniques, create at least 3 different point mutations in the gene (in vitro).
Then, using lysates of WT cells transfected with siRNA, cells we created that carry the point mutations, and cells from the knockout mice/strains (all will be the same cell type), we will extract mRNA and proteins. We will quantify mRNA levels using RT-PCR and protein levels using western blots. Microarrays against human genes will also be done using mRNA from the sample that showed the most differential expression versus the siRNA and the siRNA sample to compare off-target effects.
If it is shown that siRNA is just as effective (or potentially more effective) in silencing a gene as creating a deletion or a mutation, and it does not have more off-target effects than the other method, then siRNAs can be used for gene silencing studies. The advantage to that is that using siRNAs will be a lot easier than creating a deletion or a mutation. Also, adding the siRNA can be useful for creating temporal silencing as opposed to permanent silencing, and that can be very advantageous in numerous types of studies.
