User:Drew C. MacKellar/Notebook/ARPA-E/2011/06/24

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Entry title
KAS engineering:

1. I collected the o/n cultures of the strains Jeff ordered. I'd grown ~7mL per strain, and had to use plain LB with no antibiotics for the M5 strain, since I couldn't find any info about its resistances. I hope that the cultures didn't become contaminated, because I'm not familiar with the tubes used, and the 30C incubator is pretty raunchy (mostly smells of fungus, though, not other bacteria). I froze down 1mL aliquots of the cultures and also miniprepped them, so that I can check them by PCR if I get the relevant primers.

After checking over the papers that describe each strain, it's a little too difficult to track down all the insertions and alterations that might be present at particular loci, so I'll just have to sequence the fabB & fabF from each strain. I can do this with the primers designed for amplifying these genes, which can then be put into pUC19. Then, I can sequence across the pUC19 MCS using the M13 primers.

2. I finished designing primers for mutating FabF today, based on the residues Jeff or I picked out. The basic idea is to amplify the fabF gene (or the fabB gene or the Umbellularia californica thioesterase), and cloning into pUC19. That vector is small, and should be great for performing the point mutations using the Phusion kit. Then we will use the oligos indicated to perform the mutations; for the couple of mutants Jeff suggested that involve two mutations, each mutation will be performed as a separate step. The mutations involve the same forward primer for each residue being mutated, then a different reverse primer for each residue you want to mutate it to. The kit requires adding a 5' phosphorylation to each primer; this costs ~$20 per primer to have this added during synthesis, about doubling the cost of each primer. I think instead we'll get a kit and perform this step ourselves. Then, after sequencing with the M13 primers to confirm that we've got what we want, we can cut out with the BamHI/SalI sites and drop it into the pACYC Duet vector.

fabF Point mutants	I108M		..	3923	Tm=80.9, TaOPT=59.8, dTm=20.6

T	CGGCACCAATGCGGGTTG	18	60.3	-41.4

B	CAATTGGCTCCGGGATGGGCGGCCTCGGACTGA	33	82.1	-76.3

I108F	B	CAATTGGCTCCGGGtTTGGCGGCCTCGGACTGA	33	65.5	-72.1

I108L	B	CAATTGGCTCCGGGcTTGGCGGCCTCGGA	29	64.9	-65.6

fabF point mutants	L111M		..	3923	Tm=80.9, TaOPT=58.9, dTm=23.3

T	CGGAGCCAATTGCGGCAC	18	59.6	-41.3

B	GGATTGGCGGCaTgGGACTGATCGA	25	57.5	-45.1

L111F	B	GGATTGGCGGCtTCGGACTGATCG	24	57.4	-48.7

L111Q	B	GGATTGGCGGCCagGGACTGATCGA	25	59.1	-48.2

fabF point mutants	I114F		..	3923	Tm=80.9, TaOPT=58.2, dTm=25.8

T	AAGGCATCCATCTTGCGCTGTT	22	59.6	-44.8

B	CATTCAATATGGAtTTGTCGCTGGCGTTC	29	55	-52.9

I114K	B	CATTCAATATGGAAagGTCGCTGGCGTTC	29	56.4	-50.9

I114W	B	CATTCAATATGGAtggGTCGCTGGCGTTC	29	57.8	-49

I114M	B	CATTCAATATGGAATgGTCGCTGGCGTTC	29	56.4	-52.4

fabF point mutants	F133Y		..	3923	Tm=80.9, TaOPT=58.0, dTm=26.6

T	TCTTACGTGGACCACCGTTCATC	23	57.6	-43

B	TCAGCCCATTCTaCGTTCCGTCAAC	25	54.2	-46

F133W	B	TCAGCCCATTCTggGTTCCGTCAAC	25	55.9	-42.4

fabF point mutants	I138M		..	3923	Tm=80.9, TaOPT=57.5, dTm=28.3

T	CGAAGAATGGGCTGATCTTACGT	23	56.3	-43.7

B	TTCCGTCAACGATgGTGAACATGGT	25	52.6	-42.7

I138F	B	TTCCGTCAACGtTTGTGAACATGGT	25	50.9	-43.5

fabF point mutants	L197M		..	3923	Tm=80.9, TaOPT=58.4, dTm=25.1

T	CTTTCTCTGCGCCACCTGC	19	55.8	-39.5

B	CCAGTACGCCGaTGGGCGTTGGTGG	25	60.8	-52.9

L197F	B	CCAGTACGCCGtTtGGCGTTGGTGG	25	59.1	-47.9

Uc C12 TES	pACYC 20110623		..	924	Tm=80.8, TaOPT=58.4, dTm=25.2

T	CAGTcatatgACGAATCTCGAATGGAAACCTAA	33	55.6	-47.3

B	TTTCTctcgagGACCCGGGGTTCAG	25	57.5	-37.1

fabB pACYC 20110623	Pair 1		<GACCTAgTcgacATTAATCTTTCAGCTTGCGC>..<CCAGTAATCACTGCACGTTTCATTggATcCCTCTGTA>	1248	Tm=82.5, TaOPT=60.2, dTm=24.4

T	GACCTAgTcgacATTAATCTTTCAGCTTGCGC	32	58	-46.8

B	TACAGAGGgATccAATGAAACGTGCAGTGATTACTGG	37	60.5	-56.3

fabF pACYC 20110623	Pair 1		<CTGGAGGgatccCGTGTCTAAGCGTCGTGTAGTTGT>..<CTTTGATCTTTAAAAAGATCTAAGgTcgacTTTTCCA>	1269	Tm=81.1, TaOPT=58.4, dTm=26.2

T	CTGGAGGgatccCGTGTCTAAGCGTCGTGTAGTTGT	36	63.9	-55.6

B	TGGAAAAgtcgAcCTTAGATCTTTTTAAAGATCAAAG	37	55	-50.8