User:Justin Tan

Registration/Questionnaire: 20.109 Fall 2007

Last Name

Tan

First Name

Justin

Preferred name

Justin

Course/Minor

20 - Biological Engineering 15 - Management

Year of Graduation

2009

Telephone #

339-221-2455

Email

jtan_87 AT mit DOT edu

Have you taken

7.05/5.07 (Biochemistry) - YES 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

In high school, I did a lot of biochemistry research. When I arrived at MIT, I worked at the Langer Lab doing tissue engineering research, specifically with regards to the osteogenic stem cell line. Finally, throughout my sophomore year, I worked in the Hamad-Schifferli lab designing temperature-sensitive biomedical devices for drug delivery.

Anything else you would like us to know?

I am really excited to be in this class! Although I have already had significant experience working in lab, I would really like to diversify the types of research that I do since I haven't really found a "passion" in any of the work that I've done in the past. I am also very enthusiastic about improving my oral/writing skills since it is something that I have always struggled with in the past.

M13 Re-Engineering Ideas

Protein	Function	Re-engineering Ideas
I	Assembly	Re-engineering of PI will undoubtedly cause some very drastic changes since it works in conjunction with several other genes (PIV and PXI) in the assembly process. Perhaps by altering the C-terminals of PI (and PXI) in interacting with pIV, we can alter the channels with which the phages are secreted and analyze the potential consequences of having a larger/smaller channel on the rate of secretion.
II	Replication of DNA + strand	From 7.03, I learned of various pathways that can control promoter sequences. If the natural promoter on M13 can be altered to become inducible, the rate of M13 replication within the host bacteria could potentially be controlled. Consequently, this will also have an impact on the life-cycle of M13 | | bacteriophage.
III	Phage tail protein (5 copies)	Since PIII is responsible for the initial binding of M13 to the bacteria at the TolA of the F Pilus, if this protein-binding process can be modified to bind in more varied conditions, we can potentially construct an M13 bacteriophage that can bind to bacteria without the F Pilus factor.
IV	Assembly	In the same way as PI, re-engineering of PIV will undoubtedly cause some very drastic changes since it works in conjunction with several other genes (PI and PXI) in the assembly process. Perhaps by altering the N-terminal of PI in interacting with pI and PXI, we can alter the channels with which the phages are secreted and analyze the potential consequences of having a larger/smaller channel on the rate of secretion.
V	Binds ssDNA	Altering this gene will have a very profound effect upon the efficiency of DNA replication. I would be very interested to see the differences that might arise by changing the ratio between sequestered ssDNA and the formation of dsDNA. Perhaps we can optimize the level of phage production...
VI	Phage tail protein (5 copies)	Since PVI is only an accessory protein for PIII, we can test the stability of PIII in performing its function with decreased/increased levels of PVI production. My guess is that it works to make the process more efficient...
VII	Phage head protein (5 copies)	Alter the gene so that p8 cannot be substituted for p5- see how this affects the phage (e.g. can it still be secreted?)
VIII	Phage coat protein (2700 copies)	Since PVIII surrounds the cells protein coat, I think it would be a great idea to alter the surface charge density of the protein coat. That way, we can compare M13 to its evolutionary relatives (fd and fl) to determine whether the difference in charge densities is really a fundamental difference between them and the way they function.
IX	Phage head protein (5 copies)	I could be curious to see the effect of removing the "blunt end" entirely and replacing it with the same "rounded tip" encoded by PIII.
X	DNA replication	Altering this gene will also alter gene 2 so any alteration would affect both genes, so make any number of small modifications - see what interesting phenomena result
XI	Assembly	Modify the gene so that it is longer, hopefully resulting in a larger channel- see if this could allow multiple phages to pass through, thus making the channels more effective

M13’s Closest evolutionary relatives

M13’s closest relatives are the bacteriophages fd and fl. Although they originate from the same bacteriophage family, Inoviridae, and exhibit the same circular single-stranded-DNA components, M13 differs from fd and fl in their protein coats. Whereas fd and fl both have carboxyl groups from aspartate, M13 has an amide group from its attached asparagine. As a result, M13 has a lower charge density along its surface than its bacteriophage relatives.

”Bba_M1307 is not a standard biological part and does not belong in the registry”

The M1307 sequence has been modified to include an origin of replication from the pACYC177 sequence as well as a kanamycin resistance gene so that it can propagate on Kan(+) plates. Hence, it has obviously been biologically engineered for in-vitro substrate building. However, a unique aspect of its function exists in the presence of other phage plasmids, whereyby it adopts an indirect “helper phage” role (i.e. providing the protein coat while allowing another phage plasmid (if present) into the phage capsid/host bacteria for substrate building.