Spenser Skates: Genome Engineering Day 1

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1. The major writing assignment for this module will be a description of your M13 renovation work. Use the summary in your lab notebook to start a table on your wiki user page to organize your thoughts about the existing genome. Generate a table that lists each gene and any re-engineering ideas you have for it.

Gene Location Function Re-engineering Ideas
I 3196-4242 Assembly: Forms on the inner bacterial membrane, required for phage maturation and exit from cell Try to change its interactions with the inner bacterial membrane and p4 by changing functional groups, increase or eliminate expression to see if it affects the lytic/lysogenic character of the phage
II 8268-831 DNA Replication: Creates a nick in the double stranded phage genome to initiate replication of the + strand Modify so it nicks DNA at a different site or on the E.coli genome, create a copy so it becomes independent and modification is possible without modification of the p10 genome
III 1579-2853 Tail Protein: Necessary to cap the tail end of the phage upon exit from the cell Add an element to the protein that gives it a high affinity for a head protein so that the phage binds to other copies of itself in a line, modify to allow a different amount of DNA in the phage genome
IV 4220-5500 Assembly: Creates a channel on the bacterial outer membrane necessary for phage exit from the cell Change character so channels are only created in a certain environment,increase or eliminate expression to see if it affects the lytic/lysogenic character of the phage
V 843-1106 Binds single stranded DNA: Captures the + single stranded DNA for packaging into phage particles Change the affinity for the single stranded DNA of the phage particle, make it have an affinity for the Ecoli genome, create a different interaction with p8 to modify the coat characteristics
VI 2856-3194 Tail Protein: Caps the end of the phage protein and assists in infection of a new bacterial cell Try to make the protein specific to Ecoli cells that have a specific characteristic or only able to infect under certain conditions, add a tag to see infection progress of a cell
VII 1108-1209 Head protein: Forms "blunt" end of phage particle Add an element to the protein that gives it a high affinity for a tail protein so that the phage binds to other copies of itself in a line, add a tag to see infection progress of a cell
VIII 1301-1522 Phage coat protein: Forms the vast majority of the outer coat of a phage particle Add proteins to tag the virus such as GFP. Add surface materials that affect the chemical or physical properties of the virus particle, such as solubility or interaction with other biological materials besides E.coli
IX 1206-1304 Head protein: Forms "blunt" end of phage particle Change interaction with p7, allow binding to bacterial cells so that both ends of phage can bind with the bacterial cell wall
X 496-831 DNA Replication: Regulates the amount of double stranded DNA for packaging into phage molecules Change affinity to different parts of the phage genome, increase or eliminate activity to observe changes in number of phage created/DNA in the phage particles, create a copy so it becomes independent and modification is possible without modification of the p2 genome
XI 3916-4242 Assembly: Forms on the inner bacterial membrane, required for phage maturation and exit from cell Allow channels to open or close based on some regulating element such as temperature, increase or eliminate expression to see if it affects the lytic/lysogenic character of the phage

2. Nature often preserves functionally critical genomic elements, and evolutionary cousins can help us identify which genetic elements are disposable, which are interchangeable, and which are essential. Who are M13's closest evolutionary relatives and how do they differ from the phage you're working with?
M13 have the following virus classifiction:
Familia: Inoviridae
Genus: Inovirus
Species: Enterobacteria phage M13
The M13 virus is most closely related to f1 and d1 bacteriophage. These have about a 3% difference in base pair sequence. M13 is different from other closely related bacteriophages in its lysogenic life cycle.

3. Register for an account at the Registry for Standard Biological Parts. This site is a clearing house for engineered biological parts that can be used as substrates for building. Look up part BBa_M1307 and write a response to the following criticism: "BBa_M1307 is not a standard biological part and does not belong in the registry."
BBa_M1307 is the M13K07 genome with an addition of a bacterial origin of replication and a kanamycin resistance gene. A "part" is a basic funciton encoded by genetic material. BBa_M1307 is significantly larger than a standard part as it encodes instructions for all of the M13 bacteriophage lifecycle. It is better considered as a device, which performs some defined function, in this case continual infection of Ecoli cells. However, it is still useful because it does encode multiple biological parts within the genome. Furthermore, much of the knowledge of the function of individual parts is lost without an understanding of the interaction between parts and the rest of the M13 genome, which is essential for each of the individual parts' function.

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