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Team:UT-Tokyo/LabNote
From 2011.igem.org
Lab Note
iGEM UT-Tokyo
Parts List
| Number | Part | Content | Plasmid | Length (bp) |
|---|---|---|---|---|
| #1 | J23119 | c.Promoter (Strong) | pSB1A2 | 35 |
| #2 | J23118 | c.Promoter (Medium) | BBa_J61002 | 35 |
| #3 | B0032 | RBS | pSB1A2 | 13 |
| #5 | B0014 | d.Ter | pSB1AK3 | 95 |
| #9 | E0240 | RBS-GFP-d.Ter | pSB1A2 | 876 |
| #10 | E0040 | GFP | pSB1A2 | 681 |
| #11 | E1010 | RFP | pSB2K3 | 723 |
| #14 | I712019 | fLuc | pSB1AK8 | 1653 |
| #17 | J52008 | rLuc | pSB1AK3 | 936 |
| #20 | R0011 | lacP | pSB1A2 | 55 |
| #21 | C0012 | LacI | pSB1A2 | 1128 |
| #22 | I712074 | pT7 | pSB1AK8 | 46 |
| #23 | K145001 | T7 RNA Pol. | pSB1A2 | 2655 |
| #24 | J22106 | recAp | pSB1A2 | 192 |
| #27 | C0083 | AspA | pSB2K3 | 1518 |
| #28 | K112808 | T4 phage lysis device (no promoter) | pSB1A2 | 1785 |
| #29 | - | CheZ | pSB1AK3 | 728 |
| #30 | K117000 | Lysis gene | pSB1A2 | 144 |
| #31 | - | LexA | pSB1AK3 | 750 |
| #33 | - | sulAp | pSB1AK3 | 67 |
| #34 | - | uvrAp | pSB1AK3 | 96 |
| #35 | - | recNp | ||
| #36 | R0051 | cI-repressed promoter | pSB1A2 | 49 |
| #37 | C0051 | cI repressor (LVA tagged) | pSB1A2 | \ |
| #38 | - | RecA |
Lab Diary
For convenience sake, each part (genes, promotors, etc) is represented by consecutive numbers (e.g. #20 for lac promotor). By pointing the mouse on the part number in the construct, you can find out the details of the part.
- May
- June
- July
- August
- September
- October
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'11/9/12 (Mon)
- Colony PCR
- #20-#3-14-5-2-3-17-5
- #20-3-14-5-#2-3-17-5
- #1-#3-14-5-2-3-17-5
- #36-3-14-5-2-3-17-5-#20-3-37-5 (no colony)
- #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 (PCR failed?)
Note: #20-3-37-5 colonies on the master-plate are RED. Maybe #2-3-11-5...
Note: #36-3-14-5-2-3-17-5-#20-3-37-5-pSB1A3 PCR amplification doesn't work. Seems failed extension...
- PCR amplification
- #20-3-37-5 from Master plate
- #36-3-14-5-2-3-17-5 from 0911 MP product
Note: Only one #20-3-37-5 colony is NOT RED.
- Miniprep.
- #20
- #29 (pSB1AK3)
- #2-3-17-5
- #2-3-37-5
- BBa_J04450 (pSB1C3)
- Digest
- #36-3-14-5-2-3-17-5_ES (from PCR)
- #20-3-37-5_XP (from PCR)
- pSB1A3_EP
- pSB1C3_EP
- #20-3-37-5_XP (from MP) -> O/N
- J04450_EP -> O/N
- #2-9_XP -> O/N
- #20-3-29-5_ES -> O/N
- Gel extraction
- #20_SP
- #36-3-14-5_SP
- #2-3-17-5_EX (from O/N digests)
- #36-3-14-5-2-3-17-5_ES
- #20-3-37-5_XP (from today's PCR digests)
- Culture for Miniprep.
- #20-3-37-5 from 0911 master plate -> O/N
- Dual Luciferase Assay
- #20-3-14-5-2-3-17-5
- #1-3-14-5-2-3-17-5
- #36-3-14-5-2-3-17-5-20-3-37-5-pSB1A3
- Ligation -> O/N
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP
- #3-27-5_XP-#20_SP
- #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP
'11/9/13 (Tue)
- Colony PCR
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1A3_EP
- #36-3-14-5-2-3-17-5_ES-#20-3-37-5_XP-pSB1C3_EP (failed)
- #3-27-5_XP-#20_SP (failed)
- #36-3-29-5_ES-#20-3-37-5_XP-pSB1AK3_EP
- Culture for Luciferase Assay
- #36-3-14-5-2-3-17-5-20-3-37-5(pSB1A3, pSB1C3) (IPTG 0, 1, 10, 38, 100uM)
- #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
- #1-3-14-5-2-3-17-5 (IPTG 0uM)
Note: Seemes OD600 should NOT exceed 0.4~0.5, or intracellular luciferase will be saturated.
- Miniprep.
- #20-3-14-5-2-3-17-5
- #1-3-14-5-2-3-17-5
- #20-3-37-5 (from a non-red colony on the master plate)
- Gel ext.
- #20-3-37-5_XP (from non-red colony)
- #20-3-29-5_ES(failed)
- #2-9_XP, pSB1C3_EP
Note: #20-3-29-5 was not cut!
- Dual Luciferase Assay
- #1-3-14-5-2-3-17-5 (IPTG 0uM)
- #20-3-14-5-2-3-17-5 (IPTG 0, 1, 10, 38, 100uM)
- #36-3-14-5-2-3-17-5-20-3-37-5 (#2-3-11-5?) (IPTG 0, 1, 10, 38, 100uM)
- Sequencing preparation
- #20-3-37-5 (from a non-red colony on the master plate)
- #20-3-29-5
- #3-27-5
- #31
- Digest
- #20_E
- #3-29-5_S (from MP products) (cut check)
- Ligation
- #20_SP-#3-27-5_XP
- Transformation -> O/N
- #3-14-5-2-3-17-5
- #1-3-14-5-2-3-17-5
- #20-3-14-5-2-3-17-5 (for plate stock)
- #20-3-37-5 (from a non-red colony on the master plate) (for plasmid check)
