User:Drew C. MacKellar/Notebook/ARPA-E/2011/06/23

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Entry title
KAS engineering:

1. Jeff gave me a plate with streaks of the strains he ordered from the Yale Coli Genetic Stock Center (CGSC). They grew at 30C o/n (because several have temp-sensitive mutations), and now I need to pick single colonies and grow in LB + their respective antibiotics. I'll grow overnight, then make glycerol stocks and leave the plate at 4C.

-Note: Joe told me after I set up the cultures that integrated constructs like those present in all of these strains usually require a reduced concentration of antibiotic during growth. The typical ATCC guidelines of recommended antibiotic concentrations usually assume that your bacteria have a high-copy plasmid; a single-copy plasmid or single genomic integration of the gene conferring resistance often isn't expressed at a high enough level to neutralize the antibiotic at the concentration used for high-copy plasmids, and as a result the cells sometimes undergo mutations that increase expression or copy number in the genome, which isn't desirable for our purposes. He said that the typical procedure is to halve the concentration used for high-copy plasmids.

M5

CGSC Strain#: 5641

Strain Designation: M5     Source of Strain: Broekman & Steenbakkers

Other Designations: PC1977

Sex: F- Chromosomal Markers: &lambda-, serU84(AS), fabB15(ts)

Strain Comments:

We have not been able to trace the derivation of the parental strain, K12S

serU84(AS) was formerly called supD84

AS = amber(UAG) suppressor

ts = Temperature Sensitive

References:

Broekman, JH, JF Steenbakkers 1974. Effect of the osmotic pressure of the growth medium on fabB mutants of Escherichia coli. J. Bacteriol. 117(3):971-7.

Note: no info on resistance is given in the paper, and they don't indicate any antibiotics used. Strain PC0223 has minimal info online, and no indication of whether to grow in any antibiotics. I will simply grow in plain LB for now.

CY288

CGSC Strain#: 6356

Strain Designation: CY288     Source of Strain: J.E. Cronan

Sex: F- Chromosomal Markers: fhuA22, fabF200, zcf-229::Tn10, gyrA220(NalR), fabB15(ts), relA1, rpsL146(strR), pitA10, spoT1, T2R

Strain Comments:

zcf-229::Tn10-- cotransduces 95% with purB and 3% with pyrC.

spoT1-- In addition to the 6bp insertion, there is a C to T transition resulting in an H257Y substitution.

T2R-- : selected for resistance to phage T2

= tetracycline resistant

NalR = Nalidix.acidResist.

strR = streptomycin resist.

ts = Temperature Sensitive

References:

Garwin, J.L., A.L. Klages, J.E. Cronan 1980. Structural, enzymatic, and genetic studies of beta-ketoacylacyl carrier protein synthases I and II of Escherichia coli. J.Biol.Chem. 255:11949-11956

DC374

CGSC Strain#: 7168

Strain Designation: DC374     Source of Strain: D.P. Clark

Sex: F+ Chromosomal Markers: tyrT58(AS), zea-2222::Tn10, fadD0, mel-1

Strain Comments:

The fadD allele was originally called fadF by Overath and is now thought to be fadD and may in fact be fadD88.

tyrT58(AS) was formerly called supF58

= tetracycline resistant

AS = amber(UAG) suppressor

References:

--

JW1081

CGSC Strain#: 9029

Strain Designation: JW1081-4     Source of Strain: Keio Collection

Other Designations: JW1081

Sex: F- Chromosomal Markers: Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), &lambda-, ΔfabF759::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Strain Comments:

From the Keio Collection of single-gene knockouts constructed through a collaboration of the Inst. of Adv. Biosci. at Keio Univ., the Nara Inst. of Sci. and Technol., Japan and Purdue Univ., USA

ΔlacZ4787(::rrnB-3)-- : 4 tandem copies of the rrnB transcriptional terminator inserted by gene replacement into the region extending from near the SacII site near the N-terminus of lacZ through the promoter.

rph-1-- is a 1 bp deletion that results in frameshift over last 15 codons and has polar effect on pyrE leading to suboptimal pyrimidine levels on minimal medium.(Jensen 1993 JBact.175:3401)

Δ(araD-araB)567 was formerly called Δ araBADAH33

Δ(rhaD-rhaB)568 was formerly called Δ rhaBADLD78

ΔlacZ4787(::rrnB-3) was formerly called Δ lacZWJ16

ΔlacZ4787(::rrnB-3) was formerly called ::rrnB-4

= kanamycin resistant


 * rrnB-3 = 4 copies of rrnB inserted

References:

Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1-11

JW1082

CGSC Strain#: 11795

Strain Designation: JW1082-7     Source of Strain: Keio Collection

Other Designations: JW1082

Sex: F- Chromosomal Markers: Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), &lambda-, ΔpabC760::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Strain Comments:

From the Keio Collection of single-gene knockouts constructed through a collaboration of the Inst. of Adv. Biosci. at Keio Univ., the Nara Inst. of Sci. and Technol., Japan and Purdue Univ., USA

ΔlacZ4787(::rrnB-3)-- : 4 tandem copies of the rrnB transcriptional terminator inserted by gene replacement into the region extending from near the SacII site near the N-terminus of lacZ through the promoter.

rph-1-- is a 1 bp deletion that results in frameshift over last 15 codons and has polar effect on pyrE leading to suboptimal pyrimidine levels on minimal medium.(Jensen 1993 JBact.175:3401)

Δ(araD-araB)567 was formerly called Δ araBADAH33

Δ(rhaD-rhaB)568 was formerly called Δ rhaBADLD78

ΔlacZ4787(::rrnB-3) was formerly called Δ lacZWJ16

ΔlacZ4787(::rrnB-3) was formerly called ::rrnB-4

= kanamycin resistant


 * rrnB-3 = 4 copies of rrnB inserted

References:

Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1-11 ---

JW1794

CGSC Strain#: 9503

Strain Designation: JW1794-1     Source of Strain: Keio Collection

Other Designations: JW1794

Sex: F- Chromosomal Markers: Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), &lambda-, ΔfadD730::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Strain Comments:

From the Keio Collection of single-gene knockouts constructed through a collaboration of the Inst. of Adv. Biosci. at Keio Univ., the Nara Inst. of Sci. and Technol., Japan and Purdue Univ., USA

ΔlacZ4787(::rrnB-3)-- : 4 tandem copies of the rrnB transcriptional terminator inserted by gene replacement into the region extending from near the SacII site near the N-terminus of lacZ through the promoter.

rph-1-- is a 1 bp deletion that results in frameshift over last 15 codons and has polar effect on pyrE leading to suboptimal pyrimidine levels on minimal medium.(Jensen 1993 JBact.175:3401)

Δ(araD-araB)567 was formerly called Δ araBADAH33

Δ(rhaD-rhaB)568 was formerly called Δ rhaBADLD78

ΔlacZ4787(::rrnB-3) was formerly called Δ lacZWJ16

ΔlacZ4787(::rrnB-3) was formerly called ::rrnB-4

= kanamycin resistant


 * rrnB-3 = 4 copies of rrnB inserted

References:

Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1-11 -

JW1228

CGSC Strain#: 9113

Strain Designation: JW1228-1     Source of Strain: Keio Collection

Other Designations: JW1228

Sex: F- Chromosomal Markers: Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), &lambda-, ΔadhE748::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Strain Comments:

From the Keio Collection of single-gene knockouts constructed through a collaboration of the Inst. of Adv. Biosci. at Keio Univ., the Nara Inst. of Sci. and Technol., Japan and Purdue Univ., USA

ΔlacZ4787(::rrnB-3)-- : 4 tandem copies of the rrnB transcriptional terminator inserted by gene replacement into the region extending from near the SacII site near the N-terminus of lacZ through the promoter.

rph-1-- is a 1 bp deletion that results in frameshift over last 15 codons and has polar effect on pyrE leading to suboptimal pyrimidine levels on minimal medium.(Jensen 1993 JBact.175:3401)

Δ(araD-araB)567 was formerly called Δ araBADAH33

Δ(rhaD-rhaB)568 was formerly called Δ rhaBADLD78

ΔlacZ4787(::rrnB-3) was formerly called Δ lacZWJ16

ΔlacZ4787(::rrnB-3) was formerly called ::rrnB-4

= kanamycin resistant


 * rrnB-3 = 4 copies of rrnB inserted

References:

Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1-11 ---

JW1375

CGSC Strain#: 9216

Strain Designation: JW1375-1     Source of Strain: Keio Collection

Other Designations: JW1375

Sex: F- Chromosomal Markers: Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), &lambda-, ΔldhA744::kan, rph-1, Δ(rhaD-rhaB)568, hsdR514

Strain Comments:

From the Keio Collection of single-gene knockouts constructed through a collaboration of the Inst. of Adv. Biosci. at Keio Univ., the Nara Inst. of Sci. and Technol., Japan and Purdue Univ., USA

ΔlacZ4787(::rrnB-3)-- : 4 tandem copies of the rrnB transcriptional terminator inserted by gene replacement into the region extending from near the SacII site near the N-terminus of lacZ through the promoter.

rph-1-- is a 1 bp deletion that results in frameshift over last 15 codons and has polar effect on pyrE leading to suboptimal pyrimidine levels on minimal medium.(Jensen 1993 JBact.175:3401)

Δ(araD-araB)567 was formerly called Δ araBADAH33

Δ(rhaD-rhaB)568 was formerly called Δ rhaBADLD78

ΔlacZ4787(::rrnB-3) was formerly called Δ lacZWJ16

ΔlacZ4787(::rrnB-3) was formerly called ::rrnB-4

= kanamycin resistant


 * rrnB-3 = 4 copies of rrnB inserted

References:

Baba, T., T. Ara, M. Hasegawa, Y. Takai, Y. Okumura, M. Baba, K.A. Datsenko, M. Tomita, B.L. Wanner, H. Mori 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1-11


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