User:Amirah

protein	function / re-engineering ideas
I	assembly : alter gene so that surface receptors are present on P1 so that it can determine the number of bacteria phages being secreted at a time.
II	replication of DNA + strand : encode so that P2 can be switched on and off based on the environment which it's in, example: stops replication when in a certian concentration of Ca2+
III	phage tail protein (5 copies): possible mechanism for selectivity to only certain bacteria, dealing with P3 connectivity to the TolA protein on bacterial pilus.
IV	assembly : [look into how P3/P6 cap put on] re-engineer so that multi-phage bacterial links are formed, which requires altering the mechanism of how P4 interacts with the P3/P6 cap with which the phage is secreted.
V	binds ssDNA : alter interaction with P9/P7 so that a limited number of ssDNA maybe surrounded by P8 at a time.
VI	phage tail protein (5 copies): re-engineering closely linked with that of Gene III. Alter it to assist with bacterial selectivity.
VII	phage head protein (5 copies): alter gene so as to alter its confor- mation. A change in conformation can expand the different residues that can be attatched to its N-terminal portion.
VIII	phage coat protein (2700 copies): alter genes so that the protein P8 has an affinity for certain residues or salts. This can vastly increase the function of m13 as a whole. It can be used to transport different things into bacteria.
IX	phage head protein (5 copies): re-engineering closely linked to that of Gene VII. However, since 9 is located on the surface of m13, it can be altered so that it can express different reactive chains on the phage surface.
X	DNA replication : since altering Gene X is synonymous with altering Gene II, I would leave it alone and concentrate more on changing the functionality of gene 2. This makes more sense since Gene II as a better defined function within m13.
XI	assembly : functionality of 11 is closely linked to that of Gene 4 and Gene 1. Re-engineer so that it can assist with forming multi- phage chains, or so that it can assist with controlling replication.

Diagnostic digest 1	plasmid with insert	plasmid no insert
Enzyme(s) used	Zra1	Zra1
Buffer used	NEBuffer 2 SEBuffer B	NEBuffer 2 SEBuffer B
Temperature	37 celsius	37 celsius
Predicted fragments	8708 bp	none, circular DNA
Diagnostic digest 2	plasmid with insert	plasmid no insert
Enzyme(s) used	BamHI	BamHI
Buffer used	NEBuffer 2	NEBuffer 2
Temperature	37 celsius	37 celsius
Predicted fragments	none, circular DNA	8669 bp

Alteration	Method
Deleted Gene VII	assembly : point mutation on codon within the RBS of gene VII while still maintaining the same amino acid.
Added dual restriction enzyme buffer region	Inserted BamHI and EagI restriction sites after the end of the Gene IX orf.
Moved Gene VIII promoter	point mutation on codon within promoter region which is embedded in gene IX. acutal promotor is shifted after the buffer region.
Moved Gene VIII RBS	point mutation on the initial RBS located within Gene IX. Then shifted un-mutated RBS after new promotor region.
Removal of overlap between Gene IX and Gene VIII	Point mutation on start codon ATG to ATC which does not alter the amino acid. Start codon for Gene VIII orf then shifted to after the new RBS.
Problematic Genes	Reasoning
Gene X	Gene X is completely located within Gene II, its RBC, promoter region and ORF. I would hesistate to refactor this portion of the M13 genome because of the risk of many complications. Moving Gene X would also cause the genome to grow a considerable amount since it will have 2 copies of Gene X, its RBS, and promoter (one active, one inactive). Also, going back to functionality. Both Gene II and Gene X play a part in propagation of sDNA within the host cell, there functions must be closely linked considering the complete overlap of Gene X in Gene II.

SAGA subunits, S. cerevisiae

Ada subunits	size,chromosome,null p-type	notes
Ada1 (aka HFI1, SUP110, SRM12, GAN1)	1.467 kb/489 aa, Chr. XVI, viable
Ada2 (aka SWI8)	1.305 kb/434aa, Chr. IV, viable
Ada3(aka NGG1, SWI7)	2.109 kb/702aa, Chr. IV, viable
Gcn5 (aka ADA4, SWI9)	1.32 kb/439aa, Chr. VII, viable
Ada5 (aka SPT20)	1.815 kb/604aa, Chr. XV, viable

Spt subunits	size, chromosome, null p-type	notes
Spt3	1.014 kb/337aa, Chr. IV, viable
Spt7(aka GIT2)	3.999 kb/1332aa, Chr. II, viable
Spt8	1.809 kb/602aa, Chr. XII, viable
Spt20 (aka Ada5)	1.815 kb/604aa, Chr. XV, viable

TAF subunits	size, chromosome, null p-type	notes
TAF5 (aka TAF90)	2.397 kb/798aa, Chr. II, inviable
TAF6 (aka TAF60)	1.551 kb/516aa, Chr. VII, inviable
TAF9 (aka TAF17)	0.474 kb/157aa, Chr. XIII, inviable
TAF10 (aka TAF23, TAF25)	0.621 kb/206aa, Chr. IV, inviable
TAF12(aka TAF61, TAF68)	1.620 kb/539aa, Chr. IV, inviable

Tra1 subunit	size, chromosome, null p-type	notes
Tra1	11.235 kb/3744aa, Chr. VIII, inviable

other subunits	size, chromosome, null p-type	notes
Sgf73	1.974 kb/657aa, Chr. VII , viable
Sgf29	0.779 kb/259aa, Chr. III, viable
Sgf11	0.3 kb/99aa, Chr.XVI, viable
Ubp8	1.416 kb/471aa, Chr. XIII, viable	Forward Primer: TACTTGAAACCCTGCTTTTTTTATTTGTTATTAATAATTatgtcgaaagctacatataa Reverse Primer: ttacttttgctggccgcaTCTTTTTGTTTTATTATTATTGTTGAATGCTATTTGCTGAA
Sus1	gene with intron, Chr. II, viable

Transformation Factor	FY2068 colonies/plate
PRS416	330
no Temp PCR	0
+ Temp PCR	12

	Surface display of scFv fusion?		Binding to gold?
	Glucose	Galactose	Glucose	Galactose
pCT-CON
pAu1