Team:NTU-Taida/Notebook/Journal
From 2013.igem.org
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===Transform:=== | ===Transform:=== | ||
B0030-CinR | B0030-CinR | ||
+ | |||
RBS(B0030) | RBS(B0030) | ||
+ | |||
pCI(R0051) | pCI(R0051) | ||
+ | |||
LuxR(C0062) | LuxR(C0062) | ||
+ | |||
pLux(Lux pR, R0062) | pLux(Lux pR, R0062) | ||
+ | |||
pConst(J23119) | pConst(J23119) | ||
+ | |||
mTagBFP(K592100) | mTagBFP(K592100) | ||
+ | |||
Luciferase(J52008) | Luciferase(J52008) | ||
+ | |||
simple Las detecting system(K575024) | simple Las detecting system(K575024) | ||
+ | |||
simple Rhl detecting system(K575033) | simple Rhl detecting system(K575033) | ||
+ | |||
===Sequence:=== | ===Sequence:=== | ||
PcDFE, PcDFG, Pc ACB, Rhl B, Rhl E | PcDFE, PcDFG, Pc ACB, Rhl B, Rhl E |
Revision as of 01:39, 3 September 2013
Contents |
July
7/1~7/5
Finish RhlR and LasR circuit.
7/1
Transform:
- Positive feedback circuit:
Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G; - Control:
PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G;
(A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )
- New:
Pcin, CinR (resistant=C)
7/2
Result:
There’s no colonies at plates of Pcin and CinR.
Transform:
Pcin(resistant=A), CinR (resistant=K)
Inoculation and incubation at LB broth
- Positive feedback circuit: Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G;
- Control: PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G; (A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )
7/3
Inoculation and incubation at LB broth
Pcin(resistant=A), CinR (resistant=K)
Check
- Positive feedback circuit: Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G;
- Control: PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G; (A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )
7/4
Check and Plasmid DNA extraction (mini-prep):
- Positive feedback circuit: Pc-A-C-B, Pc-A-C-E, Pc-A-C-B, Pc-D-F-B, Pc-D-F-E, Pc-D-F-G;
- Control: PLas-B, PLas-E, PLas-G, PRhl-B, PRhl-E, PRhl-G; (A: RBS-RhlR-tt C: PRhl-RBS-RhlR D: RBS-LasR-tt F: PLas-RBS-LasR B: RBS-mCherry-tt E: RBS-GFPmut-tt G: RBS-mRFP-tt )
- Pcin(resistant=A), CinR (resistant=K)
7/5
Check: (for plasmid ready to be sequenced)
ACB-4, ACE-5, ACG-1, DFB-1, DFE-2, DFE-3, DFE-4, DFE-5, DFG-1, RhlB-1, RhlE-2, RhlG-1, RhlG-2, LasB3, LasE1, LasG4 (8,9,11 well failed :DFE-4/DFE-5/ RhlB-1)
7/8~7/12
Construct the circuit for new receptor CinR
7/8
Sequence:
ACB-4, ACE-5, ACG-1, DFB-1, DFE-2, DFE-3, DFG-1, RhlE-2, RhlG-1, RhlG-2, LasB3, LasE1, LasG4
Primer list:
Primer order | Name | sequence | |
---|---|---|---|
BMRC-120 | iGEM2013-pSB1A2 | attaccgcctttgagtgagc | R |
BMRC-153 | iGEM2013-pSB1A2 | gtgccacctgacgtctaagaa | F |
BMRC-122 | iGEM2013-mCherry | gccgtcctcgaagttcatcac | mcherry |
BMRC-124 | iGEM2013-mRFP | aacggtaacaccaccgtc | RFP |
BMRC-126 | iGEM2013-GFP | cttgtagttcccgtcatcttt | GFP |
Digestion, ligation, transform: 3A assembly and standard assembly both
B0030+CinR
7/9
Result:
The plate of B0030+CinR following 3A assembly was OK. The other following standard assembly failed unexpectedly.
Inoculation and incubation:
B0030+cinR
7/10
Plasmid DNA extraction (mini-prep):
B0030-cinR
Result:
A260/A280=1.3
Re-inoculation and incubation of plate of B0030-CinR
7/11
=Plasmid DNA extraction (mini-prep):
B0030-cinR
Result:
A260/A280=1.3
7/12
Digestion: standard assembly only:
B0030 CinR
Result:
There’s no band for B0030
7/15~7/19
CinR, LuxR
7/15
Ligation:
B0030+CinR (The certain content in the ependorf labeled B0030 undergone digestion was actually previously digested; thus, concentration was assumed to be low)
Transform:
B0030-CinR pCI CI
7/16
Result:
Only one colony of plate(B0030-CinR) grew. CI are successfully incubated. pCI failed to grow.
Conclusion:
B0030 threw away.
7/17
Digestion and ligation:
B0030 (eppendorf from early stage)
Transform:
B0030-CinR
RBS(B0030)
pCI(R0051)
LuxR(C0062)
pLux(Lux pR, R0062)
pConst(J23119)
mTagBFP(K592100)
Luciferase(J52008)
simple Las detecting system(K575024)
simple Rhl detecting system(K575033)
Sequence:
PcDFE, PcDFG, Pc ACB, Rhl B, Rhl E Total 16 tube
Sequence results:
No single plasmid was correct at all.
7/18
7/19
7/22~7/31
7/22
7/23
7/24
7/25
7/26
7/27
7/28
7/29
7/30
7/31
July
2