Team:Osaka/Notebook

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Revision as of 09:46, 13 October 2010

Calendar

July
Week	S	M	T	W	T	F	S
					1	2	3
	4	5	6	7	8	9	10
	11	12	13	14	15	16	17
	18	19	20	21	22	23	24
week1	25	26	27	28	29	30	31

August
Week	S	M	T	W	T	F	S
week2	1	2	3	4	5	6	7
week3	8	9	10	11	12	13	14
week4	15	16	17	18	19	20	21
week5	22	23	24	25	26	27	28
week6	29	30	31

September
Week	S	M	T	W	T	F	S
week6				1	2	3	4
week7	5	6	7	8	9	10	11
week8	12	13	14	15	16	17	18
week9	19	20	21	22	23	24	25
week10	26	27	28	29	30

October
S	M	T	W	T	F	S
					1	2
3	4	5	6	7	8	9
10	11	12	13	14	15	16
17	18	19	20	21	22	23
24	25	26	27	28	29	30
31

November
S	M	T	W	T	F	S
	1	2	3	4	5	6
7	8	9	10	11	12	13
14	15	16	17	18	19	20
21	22	23	24	25	26	27
28	29	30

Notebook

September 19 (Sun)

PCR of pgsB (repeat)
- again, no product :(
PCR of pgsB (4th attempt, including yesterday's)
- no product
Miniprep of 004, 005, 008, 009, 018, 019
Restriction digest of 018 with EcoRI, SpeI; 019 with XbaI, PstI
Gel electrophoresis of pgsA, 004, 005, 008, 009, 018, 019
- apart from 018 & 019, all are parts digested before; used here to compare with new parts 018, 019
- (RESULTS?)
- problem with 019?
PCR of pgsB 1st fragment (5th attempt)
- (RESULTS?)
PCR of pgsB - generation of 2nd fragment (170bp) for overlap extension
PCR of pgsB - generation of 3rd fragment (1000bp) for overlap extension
PCR: Phusion activity check using BglX as template & the primers that generated it
PCR: pgsB template check using the outermost primers

September 20 (Mon)

Transfer to solution culture: 004, 005, 006, 007, 008 ,009 ,019
PCR: Phusion polymerase & template checks (repeat of 9/19?)
- positive control (BglX) was amplified -> Phusion polymerase seems to be working ok
- problem with template? primer? thermocycle settings?
PCR: primer check
- pair of primers for each overlap segment were tested
- (RESULTS?)　
Digestion of pgsA (PCR product) and 1-1C with EcoRI, PstI
Ligation to make new part 020: pgsA as insert, 1-1C as vector
Transformation of ligation product
PCR of pgsB 1st fragment (n-th repeat??)

Note: MANY MANY rounds of PCR carried out today; due to time constraints they are not described here in detail

September 21 (Tue)

Miniprep of yesterday's cultures: 004, 005, 006, 007, 008 ,009; 019 was discarded (turned red)
Restriction digest of miniprepped parts with EcoRI, SpeI
Gel electrophoresis of digested parts 004, 005, 006, 007, 008, 009, 019 (?)
- 005 - OK
- 014 - no band visible
- 019 - bad length - repeat ligation
- 006, 007 - bad lengths; repeat colony pick-up & culture?
PCR to synthesize C-terminal half of pgsB (overlap extension method continued)
- band of correct size obtained!
PCR cloning of Man26B, CelB
- gel run failed to turn up bands; repeat with lower annealing temp
- repeat run succeeded!
Inoculated YPD liquid culture medium with yeast
New part 020 (contains pgsA) transferred to solution culture
PCR of pgsB (final step - extension of overlapping fragments)
Gel electrophoresis to extract PCR products (Man26, CelB) as well as 1-5A for plasmid backbone

September 22 (Wed)

Gel electrophoresis of 1-5A, 1-3A, 1-1C, PCR product (pgsB) followed by extraction/purification
Restriction digest of extracted parts with EcoRI, SpeI
Miniprep of 020
Restriction digest of 020 with XbaI, PstI (for gel run to check & further assembly)
Restriction digest of pgsC, pgsA with EcoRI, SpeI
Gel electrophoresis of all digested parts above
- 020 was bad; repeat ligation?
Transfer of 006, 007 to solution culture (pick up from new colonies?)
Ligations
- 019: pgsC (PCR product) into 1-1C vector
- 020: pgsA (PCR product) into 1-1C vector
- 021: pgsB (PCR product) into 1-1C vector
- all using PCR products purified today
Transformation of above ligation products
Extraction of genome DNA from yeast cultured yesterday
PCR cloning of yeast parts from genomic DNA
- ADH1 terminator
- ADH2 promoter
- CYC1 terminator
- ENO2 promoter
- SUC2 leader sequence

September 23 (Thu)

Gel electrophoresis of yesterday's PCR products followed by extraction
Restriction digest of PCR products with EcoRI, SpeI
- ENO2, ADH2 incorrect length -> repeat
PCR cloning of ENO2 promoter, ADH2 promoter, glr (glutamate racemase)
Gel electrophoresis of crude PCR product, extraction & purification from gel
- ENO2 promoter, ADH2 promoter, glr obtained!
PCR cloning of CelB, Man26B, Cel44A (Cel44A: internal mutations needed; 1st step of overlap extension fragment generation)
Gel electrophoresis of CelB, Man26B, Cel44A PCR products
- (RESULTS?)
Miniprep of 006, 007 followed by restriction digest with EcoRI, SpeI
Gel electrophoresis of 006, 007
- (RESULTS?)
Transfer to solution culture: 019, 020
- no white/non-RFP colonies on 021 (pgsB) plate
  - insert (PCR product) was not digested properly?
  - problem with gel purification?

September 24 (Fri)

Inoculation of Karita-sensei's cellulase parts into fresh LB (need more plasmid); also, 024 (beta-glucosidase)
Extraction from iGEM distribution plates:

ID	Part Name	Resistance	Description
1-6N	<bbpart>BBa_</bbpart>	A,K	T7 promoter
2-2F	<bbpart>BBa_</bbpart>	A	T7 polymerase
1-6I	<bbpart>BBa_</bbpart>	A	tetracycline-repressible promoter

PCR purification of yesterday's Cel44A
Miniprep of 019, 020
Restriction digest of 019, 020 with XbaI, PstI
- inserts of correct lengths obtained!
Ligations for 3A assembly
- 004: 001 as upstream, 1-2J as downstream, 1-3A as vector
- 005: 001 as upstream, 2-22P as downstream, 1-3A as vector
- pgsB 10xHC 1-3A ???
Transformation of ligation products
PCR cloning (repeat) of Man26, CelB
Gel electrophoresis
- (RESULTS?)

September 25 (Sat)

Miniprep of yesterday's solution cultures (cellulase parts-containing plasmids from Karita-sensei)
Restriction digest of miniprepped plasmid DNA with XbaI, PstI (??? these are not biobrick plasmids!) & gel electrophoresis
Transformation of miniprepped parts
Restriction digests
- 001 with EcoRI, PstI
- K1 (??) with EcoRI, SpeI
Ligations to transfer 001, K1 into 1-1C (Amp-resistance) vector -> designated as 001-2
- 025: xylanase (K1) in 1-1C vector
PCR cloning of CelB
Transformation of 001-2, 025

September 26 (Sun)

Transfer to culture solution (yesterday's transformations)
Miniprep of 1-6N, 2-2F, 1-6I, 021 faint hint of red detected
Restriction digest
- 1-6N, 1-6I with EcoRI, SpeI
- 2-2F, 021 with XbaI, PstI
Gel electrophoresis
- (RESULTS?)
Miniprep of parts moved to solution culture this morning
Cut check with XbaI, PstI <- ??these are not biobrick plasmids!
- Cel8 - ok
- Cel44 - ??
- Man26 - ok
- Xyn10 - ??
Ligations of 9/23 restriction digests (ADH1, ADH2, CYC1, ENO2, SUC2, glr) to 1-1C plasmid backbone digested at E, S sites
- 026 - ADH1 terminator
- 027 - ADH2 promoter
- 028 - CYC1 terminator
- 029 - ENO2 promoter
- 030 - SUC2 leader sequence
- 031 - glr (glutamate racemase)
Transformation of ligation products
Transfer of yesterday's transformations to solution culture: 001-2, 025

September 27 (Mon)

PCR of Man26, CelB
PCR of Man48 (??), CBM from XynAcc
Restriction digests
- 1-2M with EcoRI, SpeI for assembly later
- Cel8, Cel44, Cel5 with EcoRI; Xyn10 with PstI (for checking?)
- 001-2 with EcoRI, SpeI
- 025 with XbaI, PstI
Gel electrophoresis
Ligations
- 032: 1-2M as upstream, 019 as downstream, 1-3A as vector
- 033: 1-2M as upstream, 020 as downstream, 1-3A as vector
- 004: 001-2 as upstream, 1-2J as downstream, 1-3A as vector (remake using 001-2)
- 005: 001-2 as upstream, 2-22P as downstream, 1-3A as vector (" ")
Transformation of ligation products
Transfer to solution culture: 026, 027, 028, 029, 030, 031

September 28 (Tue)

Miniprep of 026, 027, 028, 030, 031
Restriction digest
- 026, 028, 031 with XbaI, PstI
- 027, 030 with EcoRI, SpeI
Gel electrophoresis
- (RESULTS?)
Ligation: repeat assemblies of 004, 005, 032, 033 yesterday
Transformation of ligation products as well as 3-2P

ID	Part Name	Resistance	Description
3-2P	<bbpart>BBa_</bbpart>	A	??

September 29 (Wed)

Transfer to solution culture 025, 026, 027, 028, 030, 031
Ligations (repeat of 9/26 with different dilution of 1-1C)
- 026 - ADH1 terminator
- 027 - ADH2 promoter
- 028 - CYC1 terminator
- 029 - ENO2 promoter
- 030 - SUC2 leader sequence
- 031 - glr (glutamate racemase)
Transformation of 025, 026, 027, 028, 030, 031 (repeat just in case; previous plates almost all red colonies; cannot pick up colony with correct insert?)

PCR
- pgsB - cloning from plasmid
- pgsB - amplification from 021
- Man28 - amplification from previous product
PCR purification of products

PCR of CelB, XynA-CBM

Miniprep of cultures inoculated this morning (total culture time: 12hr)
- 025 -> ok (colorless)
- 027 -> turned red; discarded
- others: ok?
Restriction digest of 026, 028, 031 with XbaI, PstI
Transfer to solution culture (9/28 transformations of 004, 005, 032, 033, 3-2P)
Gel electrophoresis of PCR products

Restriction digests:
- pgsB, Man (??) with EcoRI, SpeI
- today's miniprepped plasmids with EcoRI
- 1-1C with EcoRI, SpeI

September 30 (Thu)

PCR cloning
- CM10
- CelB (repeat)
- ADH1 terminator, CYC1 terminator, SUC2 leader sequence from yeast genome (repeat)
- glr (repeat)
PCR purification: CM10, XynA-CBM
Restriction digest of all above PCR products with EcoRI, SpeI
Gel electrophoresis

Ligation of PCR products to 1-1C backbone
- 021: pgsB
- 025: XynA-CBM
- 026: ADH1 terminator
- 028: CYC1 terminator
- 031: glr
- 034: Man
- 035: CM10
Transformation of ligation products

PCR cloning of CelB, SUC2, ECO2, ADH2 from genomic DNA
Gel electrophoresis
- (RESULTS?)

Miniprep of 004, 005, 032, 033, 3-2P (9/29 solution culture)
Restriction digest of miniprepped parts
- 004, 005, 032 with EcoRI, SpeI (same as earlier today)
- 033, 3-2P (same as on 9/29)
Gel electrophoresis
- (RESULTS?)

TO CHECK/CONFIRM

'K1' (mentioned on 9/250 -> where did it come from?
9/25 - miniprepped parts (inoculated 9/24) were plasmids from karita sensei? biobrick parts pcr-cloned on 9/23? why where these parts transformed immediately after miniprep?
'CelB, Cel44A' etc vs 'Cel5, Cel8, Cel44' which nomenclature refers to what (received plasmids, pcr product?)

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