Team:Hong Kong HKUST/wetlab/notebook

From 2014.igem.org

(Difference between revisions)

@@ Line 1,329: / Line 1,329: @@
 . 	ligation of TAs and CRs	<br>
 . 	Colony PCR	<br>
-. 	Digest Receptor Backbone pSB3K3	<br>
+. 	Digest Receptor Backbone pSB1C3	<br>
 . 	Agar-LB plates	<br>
 . 	Inoculation for transformed Biobricks	<br>
@@ Line 1,345: / Line 1,345: @@
 . 	Re-ligation of TAs and CRs	<br>
 . 	P<sub>bad</sub> PCR clean up and Digestion	<br>
-. 	Ligation of P<sub>bad</sub> with pSB3K3	<br>
+. 	Ligation of P<sub>bad</sub> with pSB1C3	<br>
 . 	Sequencing preparation of P<sub>bad</sub> <br>
@@ Line 1,366: / Line 1,366: @@
 <br> <b> July 11</b> <br>
 Wet lab<br>
-. 	P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB3K3 with EcoR1-HF and Spe1 and gel purification	<br>
+. 	P<sub>bad</sub> digestion and gel purification, Digestion of P<sub>bad</sub>-pSB1C3 with EcoR1-HF and Spe1 and gel purification	<br>
 . 	Ligation of P<sub>bad</sub> and BBa_E0240	<br>
 . 	Overlapping PCR design	<br>
@@ Line 1,390: / Line 1,390: @@
 . 	Ligation of P<sub>bad</sub> an BBa_E0240	<br>
 . 	Competent cells preparations for DH5alpha and DH10B	<br>
-. 	Prepare receptor backbone for oligo anealing, pSB3K3-BBa_J04450 (EcoRI, SpeI)	<br>
+. 	Prepare receptor backbone for oligo anealing, pSB1C3-BBa_J04450 (EcoRI, SpeI)	<br>
 . 	One full set of re-ligation (short time)	<br>
 . 	One full set of re-ligation (O/N)	<br>
@@ Line 1,463: / Line 1,463: @@
 . 	Gibson assembly for amilCP	<br>
 . 	Miniprep CRs TAs	<br>
-. 	restriction check pSB3K3-TAs/CRs constructs	<br>
+. 	restriction check pSB1C3-TAs/CRs constructs	<br>
 . 	Restrict GFP.dTT for CRs	<br>
 . 	Restrict P<sub>bad</sub> for TAs	<br>
@@ Line 1,470: / Line 1,470: @@
 . 	Colony PCR	<br>
 . 	inoculation of TAs and CRs	<br>
-. 	prepare P<sub>bad</sub> L(+) A. concentration gradients for characterization	<br>
+. 	prepare P<sub>bad</sub>L(+) A. concentration gradients for characterization	<br>
 . 	Restrict CRs	<br>
 . 	Restrict TAs-Version1	<br>
@@ Line 1,485: / Line 1,485: @@
 . 	Colony PCR	<br>
 . 	Miniprep CRs TAs	<br>
-. 	restriction check pSB3K3-TAs/CRs constructs	<br>
+. 	restriction check pSB1C3-TAs/CRs constructs	<br>
 . 	Restrict BBa_B0015-Version2	<br>
@@ Line 1,503: / Line 1,503: @@
 Wet lab<br>
 . 	miniprep candidate colony or construct	<br>
-. 	restriction check pSB3K3-TAs/CRs constructs	<br>
+. 	restriction check pSB1C3-TAs/CRs constructs	<br>
 . 	Re-Re-ligation transformation	<br>
 . 	Re-Re-ligation transformation V.X	<br>
@@ Line 1,535: / Line 1,535: @@
 Wet lab<br>
 . 	Miniprep and restriction check inoculations	<br>
-. 	restriction check pSB3K3-TAs/CRs constructs	<br>
+. 	restriction check pSB1C3-TAs/CRs constructs	<br>
 . 	Inoculation of CR and TA from distribution kit	<br>
-. 	prepare BBa_I20260 in pSB3K3	<br>
+. 	prepare BBa_I20260 in pSB1C3	<br>
 . 	prepare Side project Buffer	<br>
 . 	prepare side project gel red 200:1	<br>
@@ Line 1,547: / Line 1,547: @@
 . 	transform our P<sub>bad</sub> into DH5alpha	<br>
 . 	prepare M9 medium	<br>
-. 	transformation of Clon001-010 in pSB3K3 ( with a control)	<br>
+. 	transformation of Clon001-010 in pSB1C3 ( with a control)	<br>
 <br> <b> July 30</b> <br>
 Wet lab<br>
-. 	transformation of CLON001-010 in pSB3K3 (BBa_B0015, BBa_I13401)	<br>
+. 	transformation of CLON001-010 in pSB1C3 (BBa_B0015, BBa_I13401)	<br>
 . 	C. PCR for yesterday transformations (CLON001-010)	<br>
 . 	If construct after gel check inoculation, restriction, ligation	<br>
-. 	prepare BBa_I20260 in pSB3K3	<br>
+. 	prepare BBa_I20260 in pSB1C3	<br>
 . 	transform our P<sub>bad</sub>into DH5alpha	<br>
 . 	prepare M9 for characterization	<br>
@@ Line 1,570: / Line 1,570: @@
 . 	colony PCR for 5th	<br>
 . 	inoculate candidates	<br>
-. 	prepare BBa_I20260 in pSB3K3	<br>
+. 	prepare BBa_I20260 in pSB1C3	<br>
@@ Line 1,605: / Line 1,605: @@
 . 	Digest 005 Spe1 and Pst1	<br>
 . 	ligate CLON 005 with BBa_B0015	<br>
-. 	prepare backbone : BBa_B0015 E, X /// BBa_I13401 E,X /// BBa_J23102 X, P	<br>
+. 	prepare backbone : BBa_B0015 E, X /// BBa_I13401 E,X /// J23102 X, P	<br>
 . 	Ligate P<sub>bad</sub> with Key1 dTT	<br>
 . 	digest P<sub>tet</sub> lock 1 with S and P	<br>
@@ Line 1,664: / Line 1,664: @@
 . 	Glycerol stock for 001-010	<br>
 . 	Inoue competent cells	<br>
-. 	"Mini Prep pSB3K3-PCRP<sub>bad</sub><br>
+. 	"Mini Prep pSB1C3-PCRP<sub>bad</sub><br>
 <br><b>Aug 6</b> <br>
@@ Line 1,839: / Line 1,839: @@
 . 	Inoculate candidates	<br>
 . 	Prepare pSB3K3	<br>
-. 	Prepare pSB3K3 for oligo ligation	<br>
+. 	Prepare pSB1C3 for oligo ligation	<br>
 . 	Prepare BBa_I13401 for oligo ligation	<br>
 . 	Prepare BBa_B0015 for oligo ligation	<br>
@@ Line 1,853: / Line 1,853: @@
 . 	miniprep possible candidates	<br>
 . 	Restrict TA.dTT	<br>
-. 	pSB3K3-BBa_I13401 for oligo ligation	<br>
+. 	pSB1C3-BBa_I13401 for oligo ligation	<br>
 . 	BBa_B0015 for oligo ligation	<br>
-. 	pSB3K3-BBa_J04450 for oligo ligation	<br>
+. 	pSB1C3-BBa_J04450 for oligo ligation	<br>
 . 	Inoue competent cells	<br>
 . 	P<sub>bad</sub>-key3-dTT restriction check	<br>
@@ Line 1,863: / Line 1,863: @@
 . 	Gel check for P<sub>bad</sub>.TA.dTTs	<br>
 . 	purification for BBa_B0015, BBa_I13401	<br>
-. 	pSB3K3-BBa_J04450 for oligo ligation	<br>
+. 	pSB1C3-BBa_J04450 for oligo ligation	<br>
 . 	Gel check for PCR products (19/08)	<br>
-. 	PCR check for BBa_J23102, LOCK 3 GFP DTT	<br>
+. 	PCR check for J23102, LOCK 3 GFP DTT	<br>
 . 	Restriction check for key1 DTT	<br>
@@ Line 1,879: / Line 1,879: @@
 . 	Glycerol stocks	<br>
 . 	transform HKUSTlock.GFP.dTT	<br>
-. 	Transform pSB3K3-HKUSTlock	<br>
+. 	Transform pSB1C3-HKUSTlock	<br>
@@ Line 1,885: / Line 1,885: @@
 Wet lab<br>
 . 	competent cells	<br>
-. 	Colony PCR for pSB3K3-HKUSTlock1	<br>
+. 	Colony PCR for pSB1C3-HKUSTlock1	<br>
 . 	Colony PCR for HKUSTlock1.GFP.dTT	<br>
 . 	miniprep pSB3K3-J04550 for pSB3K3 Backbone	<br>
@@ Line 1,896: / Line 1,896: @@
 . 	extraction of pSB3K3	<br>
 . 	inoculate HKUSTlock1.GFP.dTT	<br>
-. 	inoculate pSB3K3-HKUSTlock1	<br>
+. 	inoculate pSB1C3-HKUSTlock1	<br>
@@ Line 1,902: / Line 1,902: @@
 Wet lab<br>
 . 	Gel check for TAs	<br>
-. 	colony PCR for UST lock in BBa_I13401 and pSB3K3	<br>
+. 	colony PCR for UST lock in BBa_I13401 and pSB1C3	<br>
 . 	transform ust key	<br>
 . 	Check competent cells	<br>
@@ Line 1,946: / Line 1,946: @@
 . 	make SOC	<br>
 . 	single cut check for TAs	<br>
-. 	make DH5alpha cell	<br>
+. 	make 5 alpha cell	<br>
@@ Line 1,956: / Line 1,956: @@
 . 	gel check for o/n PCR	<br>
 . 	restriction check for ust key 1	<br>
-. 	restrick check for key 1	<br>
+. 	restriction check for key 1	<br>
 . 	P<sub>bad</sub> for ust key 1	<br>
-. 	extract ust lock 1 for BBa_J23102	<br>
+. 	extract ust lock 1 for j23102	<br>
 . 	ligate ust lock and key	<br>
 . 	transform ust lock and key	<br>
 . 	prepare plates for macroscope	<br>
-. 	prepare DNA for sequencing	<br>
+. 	prepare dna for sequencing	<br>
 . 	SOB	<br>
 . 	SOC	<br>
@@ Line 2,010: / Line 2,010: @@
 . 	transform MCR/MTA	<br>
 . 	Glycerol stock for locks	<br>
-. 	PCR check for biobrick key1	<br>
+. 	PCR check for Biobrick key1	<br>
@@ Line 2,056: / Line 2,056: @@
 . 	Colony PCR for CR.TAs (orthogonality)	<br>
 . 	Colony PCR for sherLock-GFP-DTT	<br>
-. 	restriction check for Lost Your Key in pSB3K3	<br>
+. 	restriction check for Lost Your Key in pSB1C3	<br>
 . 	transform P<sub>bad</sub>.lost your key.DTT	<br>
 . 	inoculate orthogonality candidates	<br>
@@ Line 2,074: / Line 2,074: @@
 . 	inoculation of B0034-GFP-DTT	<br>
 . 	inoculation of Sherlock-GFP-DTT	<br>
-. 	sequencing for sherlock in pSB3K3	<br>
+. 	sequencing for sherlock in pSB1C3	<br>
 . 	digestion of B0034-GFP-DTT	<br>
 . 	digestion of sherlock-GFP-DTT	<br>
@@ Line 2,085: / Line 2,085: @@
 . 	digestion of P<sub>bad</sub> MTA dTT	<br>
 . 	digestion of P<sub>bad</sub> dTT	<br>
-. 	digestion of BBa_J23102 MCR GFP dTT	<br>
+. 	digestion of J23102 MCR GFP dTT	<br>
-. 	digestion of BBa_J23102 BBa_E0240(B0032 GFP dTT)	<br>
+. 	digestion of J23102 BBa_E0240(B0032 GFP dTT)	<br>
 . 	digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450)	<br>
-. 	Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT	<br>
+. 	Ligation of J23102 MCR GFP dTT with P<sub>bad</sub> dTT	<br>
-. 	Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT	<br>
+. 	Ligation of J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT	<br>
-. 	Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> dTT	<br>
+. 	Ligation of J23102 BBa_E0240 with P<sub>bad</sub> dTT	<br>
 . 	Plasmid PCR for SherLock-GFP-DTT	<br>
 . 	ligation of B0034-GFP-DTT	<br>
@@ Line 2,113: / Line 2,113: @@
 Wet lab<br>
 . 	Gel purification of digested insert and backbone	<br>
-. 	Ligation of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT	<br>
+. 	Ligation of J23102 MCR GFP dTT with P<sub>bad</sub> dTT	<br>
-. 	Ligation of BBa_J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT	<br>
+. 	Ligation of J23102 BBa_E0240 with P<sub>bad</sub> MTA dTT	<br>
-. 	digestion of BBa_J23102 BBa_E0240(B0032 GFP dTT)	<br>
+. 	digestion of J23102 BBa_E0240(B0032 GFP dTT)	<br>
 . 	Transformation of ligated projects mentioned above	<br>
 . 	Colony PCR for sherLock-GFP-DTT	<br>
@@ Line 2,131: / Line 2,131: @@
 <br><b>Sept 5</b> <br>
 Wet lab<br>
-. 	miniprep/glycerol stock	<br>
+. 	miniprep/ glycerol stock	<br>
 . 	Transform P<sub>bad</sub> dTT	<br>
-. 	Transform ligated produce of BBa_J23102 MCR GFP dTT with P<sub>bad</sub> dTT	<br>
+. 	Transform ligated produce of J23102 MCR GFP dTT with P<sub>bad</sub> dTT	<br>
@@ Line 2,321: / Line 2,321: @@
 <br><b>Sept 24</b> <br>
 Wet lab<br>
-. 	Competent cells preparations!	<br>
+. 	Competent cells preparations	<br>
 . 	minipreps	<br>
 . 	colony PCR	<br>
-. 	miniprep for biobrick submission	<br>
+. 	miniprep for Biobrick submission	<br>
 . 	send plasmid for sequencing	<br>
@@ Line 2,342: / Line 2,342: @@
 . 	Gel check PvuII digestions	<br>
 . 	Gel check AseI digestions	<br>
-. 	3A assembly for non pSB3K3	<br>
+. 	3A assembly for non pSB1C3	<br>
 . 	final batch of ligation for all missing component	<br>
 . 	prepare DNA for submission	<br>
@@ Line 2,367: / Line 2,367: @@
 <br><b>Sept 28</b> <br>
 Wet lab<br>
-. 	Extract pSB3K3	<br>
+. 	Extract pSB1C3	<br>
 . 	Extract 036	<br>
 . 	miniprep "H" series candidates	<br>
@@ Line 2,392: / Line 2,392: @@
 . 	restriction check AseI	<br>
 . 	restriction check NcoI	<br>
-. 	mini prep 6 + MCR/MTA (thanks from doyle)	<br>
+. 	mini prep 6 + MCR/MTA (thanks from Doyle)	<br>
 . 	Restriction check for MCR/MTA in pSB3K3.	<br>
 . 	restrict 036	<br>
@@ Line 2,398: / Line 2,398: @@
 . 	ligate monkey with sherlock	<br>
 . 	Gel check for NcoI and AseI	<br>
-. 	Get pSB3K3 backbone EcoRI and PstI	<br>
+. 	Get pSB1C3 backbone EcoRI and PstI	<br>
 . 	Fluorescence measurement of MCR/MTA for characterization (second trial)	<br>
 . 	inoculate Lock and Keys for crude functional assay.	<br>
@@ Line 2,409: / Line 2,409: @@
 . 	miniprep 11 tubes in shaker c	<br>
 . 	restriction check H" series with NcoI	<br>
-. 	Restrict inoculated colonies with AseI	<br>
+. 	Restrict inoculated colonies wiht AseI	<br>
-. 	Restrict inoculated colonies with NcoI	<br>
+. 	Restrict inoculated colonies wiht NcoI	<br>
 . 	Gel purifications	<br>
 . 	ligation of H series and some from other series...	<br>
@@ Line 2,419: / Line 2,419: @@
 Wet lab<br>
 . 	digestion of key3,HKUSTkey?	<br>
-. 	miniprep final constructs	<br>
+. 	miniprep- final constructs	<br>

Revision as of 14:51, 17 October 2014

Home
Pneumosensor
Riboregulator
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Human Practice
Team
Wetlab
Achievements
- Medal Requirements

Notebook

Throughout the course of our project, we have continuously recorded our daily work, such as wetlab activities and research findings. The notebook for Project Pneuumonsensor and Project Riboregulator are displayed separately.

Pneumosensor

June 2014

Week 1

June 9
Wet lab
· Transformation of pSB1C3-BBa_E0240, pSB1C3-BBa_B0032 and pSB1C3-BBa_K20600
· Transformation of BBa_B0034 and BBa_E0040 into DH10B

June 10
Wet lab
· Found reference promoter J23101 in BBa_I20260 from part registry
· Looked for P_combox gene sequence from the genomic DNA
· Bacterial inoculation of BBa_B0034 and BBa_E0040

June 11
Wet lab
· Looked for P_combox gene sequence from the genomic DNA
· Studied the Characteristic, function and mechnism of how promoters work
· Miniprep of BBa_B0034 and BBa_E0040

June 12
Wet lab
· Looked for P_combox gene sequence from the genomic DNA
· research on gene sequence of PcomX, how to quantify p~comE, range of concentration of p~comE to use for characterization
· explain of scar

June 13
Wet lab
· Looked for P_combox gene sequence from the genomic DNA
· research on gene sequence of PcomX, how to quantify p~comE, range of concentration of p~comE to use for characterization

Week 2

June 16
Wet lab
· Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P_combox gene sequence
· Found a possible P_combox
· Transformation of BBa_J04450 and BBa_I20260 into DH10B
· Bacterial inoculation of BBa_E0240
·

June 17
Wet lab
· Blast the gDNA of NCTC7465 strain with various strain of S. pneumoniae to look for P_combox gene sequence
· Found a possible P_combox
· Searched for other possible P_combox sequences
· Miniprep of BBa_E0240
· Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260
·

June 18
Wet lab
· Designed primers for the P_combox sequences
· Received a P_combox sequence from Wellcome Trust Sanger Institute
· Miniprep of BBa_J04450(CHL) and BBa_I20260
· Bacterial inoculation of BBa_J04450(CHL) and BBa_I20260
· Digestion of BBa_J04450(CHL) and BBa_I20260
· Gel electrophoresis of digested products
·

June 19
· Digestion of pSB1C3-BBa_E0240 at EcoRI and SpeI
· Miniprep of BBa_J04450(CHL) and BBa_I20260
· Digestion of BBa_J04450(CHL)
· Gel electrophoresis of digested product
· Bacterial inoculation of BBa_J04450(CHL)
·

June 20
· Amended primers of P_combox sequences
· Miniprep of BBa_J04450(CHL)
· Digestion of BBa_J04450(CHL)
· Gel electrophoresis of digested product
· Designed primers for cloning comD and comE

Week 3

June 23
Wet lab
· Transformation of BBa_B0015 from iGEM Kit Plate 2011 and BBa_K880005
· Primer design for PCR of comX-myc and comW-FLAG from genomic DNA of S. Pneumoniae NCTC 7465 strain
· Designed plasmid construct for P_combox
· Bacterial inoculation of BBa_J04450(CHL)
·

June 24
Wet lab
· Inoculation of BBa_B0015
· Designed new reverse primer for 100bp, 150bp, 160bp, 180bp and 249bp P_combox
· Designed forward and reverse oligos of 67bp P_combox
· Transformation of pSB1C3-BBa_I13504
· Miniprep of BBa_J04450(CHL)
· Transformation of BBa_J04450(KAN)
· Bacterial inoculation of BBa_E0240
·

June 25
Wet lab
· Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification
· Inoculation of BBa_K880005
· Inoculation of pSB1C3-BBa_E0240
· Miniprep of BBa_E0240
· Digestion of BBa_E0240
· Transformation of BBa_J04450(KAN)
· Bacterial inoculation of BBa_J04450(CHL)
·
·

June 26
Wet lab
· Digestion of BBa_K880005 plasmid extraction product with SpeI and PstI, gel extraction and gel purification
· Digestion of pSB1C3-BBa_E0240 at XbaI and PstI
· Gel electrophoresis of digested BBa_E0240
· Gel extraction and purification of digested BBa_E0240 to obtain the insert
· Miniprep of BBa_J04450(CHL)
· Digestion of BBa_J04450(CHL)
· Gel electrophoresis of digested BBa_J04450(CHL)
· Gel extraction and purification of digested BBa_J04450(CHL) to obtain pSB1C3
· Bacterial inoculation of BBa_K880005
·

June 27
Wet lab
· Ligation of BBa_B0015 insert cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI as negative control
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of pSB1C3-BBa_E0240
· Miniprep of BBa_K880005
·

Week 4

June 30
Wet lab
· Digestion of BBa_B0015 plasmid extraction product with XbaI and PstI, gel extraction and gel purification
· Gel check of purified BBa_B0015 insert
· Transformation of ligated pSB1C3-K880005.B0015
·

July 2014

Week 1

July 2
Wet lab
· Arrival of primers order for comX-myc and comW-FLAG
· Dilution of primers and design of experiment for both comX-myc and comW-FLAG
· Inoculation of ligated pSB1C3-K880005.B0015
· Inoculation of pSB3K3-BBa_J04450 and pSB1C3-BBa_E0240
· Oligo annealing for PcomX
· Bacterial inoculation of BBa_J04450 (KAN) (6 tubes), BBa_E0240 (CAM) (6 tubes), BBa_B0015 (KAN) (3 tubes)
· PCR for cloning comD and comE

July 3
Wet lab
· PCR of comX-myc using Taq DNA Polymerase with ThermoPol Buffer to check whether primers are functional
· Gel check for comX-myc PCR product
· PCR of comX-myc using Phusion High-Fidelity PCR Master Mix with HF Buffer
· Gel check for comX-myc PCR product
· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 with XbaI and PstI
· Received and stored the new common reverse primers for P_combox and sense oligo and anti-sense oligo of 67bp P_combox
· Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of digested BBa_E0240 and BBa_J04450
· Inoculation of pSB3K3-BBa_J04450
· PcomX Transformation
· PcomCDE Oligo annealing, ligation, transformation
· J04450 Plasmid Extraction
· E0240:Plasmid extraction ; Digestion; Gel electrophoresis; Gel extraction ; Gel purification
· B0015: Plasmid extraction ; Nanodrop
· comE^D58E: PCR
·

July 4
Wet lab
· PCR of comX-myc using Vent DNA Polymerase, PCR clean up
· Digestion of pSB1C30-BBa_E0240 and pSB3K3-BBa_J04450 at XbaI and PstI
· comE^D58E :Phusion PCR
· E0240: Gel purification
·

Week 2

July 7
Wet lab
· Digestion of comX-myc with XbaI and PstI
· Gel check of digested comX-myc, digestion clean up
· PCR of comW-FLAG using Vent DNA Polymerase
· Gel check for comW-FLAG PCR product, PCR clean up
· Dephosporylation of digested pSB1C3-K880005 backbone cut with SpeI and PstI
· Gel purification of digested BBa_E0240 and BBa_J04450
· Submitted primers for Gibson assembly of pSB3K3-100bpP_combox
· Inoculation of pSB3K3-BBa_J04450
· E0240 - Bacterial inoculation
· pSB1C3-PcomX: Bacterial inoculation
· pSB1C3- PcomCDE: Bacterial inoculation, Streaking
· comE^D58E :PCR cleanup; Digestion; Ligation with pSB1C3; Transformation
·

July 8
Wet lab
· Ligation of comX-myc cut with XbaI and PstI into pSB1C3-K880005 backbone cut with SpeI and PstI
· 2nd attempt of ligation of BBa_B0015 insert cut with XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut with SpeI and PstI as negative control
· Transformation of ligation products
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of digested BBa_J04450
· Received pCEPcin from Universite de Toulouse, France
· J04450 (KAN): Digestion; Gel electrophoresis; Gel extraction
· E0240: Plasmid extraction
· pSB1C3-PcomX: Plasmid extraction
· pSB1C3- PcomCDE: Plasmid extraction
· comE^D58E: Bacterial inoculation; Ligation; Transformation
·

July 9
Wet lab
· Inoculation of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc
· Ethanol precipitation of pCEPcin
· Gel purification of digested BBa_E0240
· Gel check for BBa_E0240 insert and pSB3K3 backbone
· PCR of 100bp P_combox using Phusion polymerase
· Inoculation of pCEPcin
· E0240-PcomX & PcomCDE: Digestion w/ EcoRI and PstI; Gel electrophoresis
· J04450 (KAN): Gel purification
· comE^D58E:Plasmid extraction; NanoDrop; Gel electrophoresis (not comE); Bacterial inoculation

July 10
Wet lab
· Another PCR of comW-FLAG using Vent DNA Polymerase
· Gel check for comW-FLAG PCR product, PCR clean up
· Restriction digestion check on some candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt) and ligated pSB1C3-K880005.comX-myc
· Gel check and insert wanted present in several candidate colonies of ligated pSB1C3-K880005.B0015 (2nd attempt), but no insert wanted present in any candidate colonies of ligated pSB1C3-K880005.comX-myc
· Digestion of pCEPcin at EcoRI and SpeI, Xho and BamHI
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· PCR of 100bp P_combox using Phusion polymerase
· Ethanol precipitation of BBa_E0240 insert
· Ligate E0240-PcomX & PcomCDE
· Extract J04450 (KAN)
· Restrict check & run mutagenesis comE^D58E

July 11
Wet lab
· Digestion of comW-FLAG with XbaI and PstI, digestion clean up
· Gel check for the PCR product obtained on July 10
· Digestion of pCEPcin at NcoI and XhoI
· Ethanol precipitation of pCEPcin
· Extract J04450 (KAN)
· Ligate pSB1C3 with comE^D58E
· TransformPcomCDE
·

Week 3

July 14
Wet lab
· Ligation of comW-FLAG cut on XbaI and PstI into dephosporylated pSB1C3-K880005 backbone cut on SpeI and PstI
· Transformation of ligation products
· Inoculation of ligated dephosporylated pSB1C3-K880005.comX-myc
· Digestion of pCEPcin at NcoI and XhoI
· PCR of 100bp P_combox using Phusion polymerase
· Gel purification of digested BBa_J04450
· Inoculation of pCEPcin
· PCR of comE^D58E with only 1 primer → check whether the primers are working or not
· Ligation of pSB1C3-PcomX
· Inoculation of pSB1C3-PcomCDE
· Digest B0015
· Digest comE^D58E
· Ligation of PCR product comE to pSB1C3

July 15
Wet lab
· Digestion check of ligated pSB1C3-K880005.comX-myc
· Digestion of pSB1C3-K880005.comX-myc on SpeI and PstI, but no insert wanted present
· PCR of comX using Vent DNA Polymerase, PCR clean-up
· Colony PCR of pSB1C3-K880005.comX-myc using Taq DNA Polymerase
· Gel check of pSB1C3-K880005.comX-myc PCR product (no PCR product present)
· Inoculation of dephosporylated pSB1C3-K880005.comX-myc, dephosporylated pSB1C3-K880005.comW-FLAG
· Ethanol precipitation of pCEPcin
· Gel check of the PCR product obtained on July 14
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· PCR of 150bp, 160bp, 180bp, 249bp and 300bp P_combox using Phusion polymerase
· Plasmid extraction of pSB1C3-PcomCDE
· Bacterial inoculation (5 for PcomCDE, 4 for PcomX)
· Bacterial inoculation of B0015 (6 tubes)
· Extract gel B0015 & comE^D58E
· K880005 dephosphorylation; Digestion
·

July 16
Wet lab
· Digestion check of ligated pSB1C3-K880005.comW-FLAG
· Colony PCR of pSB1C3-K880005.comW-FLAG
· Gel check of pSB1C3-K880005.comW-FLAG PCR product (no PCR product present)
· PCR of comW using Vent DNA Polymerase (no PCR product present)
· Digestion of comX on XbaI and PstI
· Gel check of the PCR product obtained on July 15
· Gel purification of digested pSB3K3 backbone
· Digestion of pCEPcin2 at XhoI and SalI
· PCR of 150bp, 160bp, 180bp, 249bp and 300bp P_combox using Phusion polymerase
· Inoculation of pCEPcin2 and pSB3K3-BBa_I20260
· Plasmid extraction of Promoters and B0015
· Colony PCR
· Extract gel B0015 & comE^D58E
· Mutagenesis attempts for comE

July 17
Wet lab
· PCR of comX-myc and comX using Vent DNA Polymerase
· Gel check of comX-myc and comX PCR product, PCR clean-up
· PCR of comW using Vent DNA Polymerase
· Gel extraction and gel purification of comW PCR product
· Dephosporylation of BBa_K880005
· Gel check of the PCR product obtained on July 16, all types of P_combox shown positive result
· PCR clean-up of P_combox
· PCR product ligation with pSB1C3
· Bacterial inoculation of B0015, comE^D58E
· Extract gel B0015 & comE^D58E - Digestion
· Overnight Ligation
·

July 18
Wet lab
· PCR of comX using Vent DNA Polymerase
· Gel check of comX PCR product, PCR clean-up
· Digestion of comX PCR product on XbaI and PstI, digestion clean-up
· Digestion of comX-myc and comW PCR products, digestion clean-up
· Gel extraction and gel purification of digested comX and comX-myc
· Digestion of pCEPcin2 (at XhoI and SalI) and pSB3K3-BBa_I20260 (at XbaI and PstI)
· Digestion of pCEPcin2 (at BamHI and NcoI)
· PCR of 100bp P_combox using Phusion polymerase
· Gel check of the purified P_combox obtained July 17
· Gel check of the 100bp P_combox (PCR product)
· Gel purification of digested pSB3K3 backbone
· Ligation of comE^D58E
· Gel extract comE^D58E
· BBa_B0015 Gel purification
·

Week 4

July 21
Wet lab
· Gel purification of comX and comX-myc
· Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio
· Ligation of comW-FLAG into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio
· Ligation of comX into dephosporylated pSB1C3-K880005 backbone with 1:3 and 1:5 ratio
· Ligation of comW into dephosporylated pSB1C3-K880005 backbone with 1:4, 1:7, 1:10 ratio
· Colony PCR of pSB1C3-K880005.comW-FLAG using Taq DNA Polymerase
· PCR of 100bp and 150bp P_combox using Phusion polymerase
· Gel purification of digested pSB3K3 backbone
· Colony PCR of Promoters

July 22
Wet lab
· Colony PCR of comX, comW, comX-myc, comW-FLAG and use BBa_J04450 as the positive control using Taq DNA Polymerase (no PCR product present)
· Inoculation of comX, comW, comX-myc, comW-FLAG
· Gel check of the PCR product obtained on July 21
· Oligo annealing of 67bp P_combox
· Digestion of pSB3K3-BBa_I20260 at XbaI and PstI
· PCR of 100bp,150bp and 160bp P_combox using Phusion polymerase
· Gel electrophoresis of colony PCR results
· Mutagenesis of comE^D58E
·

July 23
Wet lab
· Digestion check of comX, comW, comX-myc, comW-FLAG colony PCR products on XbaI and SpeI
· Inoculation of comW-FLAG
· Gel check of PCR product obtained on July 22
· Gel purification of digested pSB3K3 backbone
· PCR of 100bp,150bp and 160bp P_combox using Phusion polymerase
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel purification of digested pSB3K3 backbone
· Gel electrophoresis of colony PCR results
· Mutagenesis of comE^D58E
·

July 24
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and SpeI-HF
· Ligation of comW-FLAG into dephosporylated pSB1C3-K880005 backbone
· Ligation of comX-myc into dephosporylated pSB1C3-K880005 backbone
· PCR of comX-myc and comX using Vent DNA Polymerase, gel extraction and gel purification
· Digestion of comX-myc and comX on XbaI and PstI-HF
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel Check of PCR product obtained on July 23
· Received pCEPcin2
· Gel electrophoresis of colony PCR and mutagenesis results
·

July 25
Wet lab
· Colony PCR of comX-myc using Taq DNA Polymerase
· Gel check of comX-myc PCR product (no PCR product present)
· Gel purification of digested pSB3K3 backbone
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Gel check on K880005
· Transformation of ligated product (promoters+pSB1C3)
·

Week 5

July 28
Wet lab
· Colony PCR of comX-myc using Taq DNA Polymerase
· Colony PCR of comW-FLAG using Taq DNA Polymerase
· Inoculation of comX-myc and comW-FLAG
· Digestion of pCEPcin2 at EcoRI and PstI, EcoRI and SpeI, NcoI and XhoI
· Colony PCR of promoters-pSB1C3
· Bacterial inoculation of K880005, comE^D58E
·
·

July 29
Wet lab
· Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF
· Transformation of comX-myc and comW-FLAG
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Digestion of pCEPcin2 at EcoRI and PstI
· Inoculation of pSB3K3-BBa_J04450
· PCR of 100bp,150bp and 160bp P_combox using Vent polymerase
· Restrict check K880005
· Promoters colony PCR
· PCR for comE fragments (mutagenesis) for Gibson Assembly

July 30
Wet lab
· Digestion check of comX-myc and comW-FLAG
· Inoculation of comX-myc and comW-FLAG
· Streaking plates of comX-myc and comW-FLAG
· PCR clean-up of 100bp,150bp and 160bp P_combox
· Digestion of pCEPcin2 at EcoRI and PstI
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· Restrict check K880005

July 31
Wet lab
· Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF
· Redigest comW-FLAG on XmnI and NcoI
· PCR sequencing for comW and comX
· Ethanol precipitation for comW and comX
· Digestion of pSB3K3-BBa_J04450 at XbaI and PstI
· K880005 dephosphorylation & ligation
· Promoters Colony PCR + Sequencing reaction
· PCR for comE^D58E fragments (mutagenesis) for Gibson Assembly

August 2014

Week 1

Aug 1
Wet lab
· Digestion of pSB1C3-BBa_J04450
· Restrict check Promoters & Sequencing reaction
· Transform K880005
·

Week 2

Aug 4
Wet lab
· Digestion of comX-myc on EcoRI-HF and SpeI, gel extraction and gel purification
· Ligation of BBa_B0015 cut on XbaI and PstI into pSB1C3-comX-myc backbone cut on SpeI and PstI
· Transformation of pSB1C3-comX-myc.B0015 ligation product
· Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
· Transformation of pSB1C3-B0015.comX-myc ligation product
· Sequencing of comX-myc and comW-FLAG
· Inoculation of comX-myc
· Digestion of 100bp, 150bp, 160bp and 180bp P_combox at XbaI and PstI
· Ligation of 100bp and 160bp P_combox with pSB1C3
· Digestion of pCEPcin2 at EcoRI and PstI
· PcomCDE Precipitation
· PcomX Sequencing reaction & precipitation
· Transform comE^D58E
· Gibson Assembly for comE and comE^D58E fragments

Aug 5
Wet lab
· Sequencing result failed
· Transformation of pSB1C3-B0015.comX-myc ligation product failed
· Digestion check of comX-myc on StyI
· Colony PCR of comX-myc
· Inoculation of comX-myc
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Ligation of 150bp, 180bp and 249bp P_combox with pSB1C3
· Transformation of pSB1C3-150bp P_combox , pSB1C3-180bp P_combox and pSB1C3-249bp P_combox
· Gel purification of digested pSB1C3 backbone
· Inoculation of pCEPcin2
· Send to sequencing: PcomCDE and PcomX
· Bacterial inoculation of comE^D58E
· Colony PCR to check pSB1C3-comE and pSB1C3-comE^D58E

Aug 6
Wet lab
· Digestion check of comX-myc on XbaI and PstI
· Gel check of comX-myc PCR product (no PCR product present)
· Sequencing of comW-FLAG using VF2 and VR
· Digestion of comX-myc on StyI
· Digestion of pCEPcin2 at EcoRI and EcoRV, EcoRI and PstI, EcoRI and SpeI, XhoI and NcoI
· Gel purification of digested pSB1C3 backbone
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Phosphorylation of 67bp P_combox oligos
· Oligo annealing for 67bp P_combox
· Align sequence with expected: P_comCDE and P_comX

Aug 7
Wet lab
· Digestion of comX-myc on StyI
· Digestion check of comX on SphI and NcoI
· Digestion check of comW on XmnI and NcoI
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Colony PCR of 100bp, 150bp, 160bp, 180bp and 249bp P_combox
· Gel purification of digested pSB1C3 backbone
· Precipitate after sequencing reaction PcomCDE
· Make glycerol stock for PcomX
· Run colony PCR for comE^D58E
·

Aug 8
Wet lab
· Digestion of comW-FLAG on EcoRI-HF and SpeI, gel extraction and gel purification
· Digestion of comW-FLAG on SpeI and PstI-HF, digestion clean-up
· Sequencing of comX-myc succeeded but mutation at EcoRI site
· Digestion check of comX-myc at EcoRI-HF and PstI-HF
· Ligation of comX and comW into dephosporylated pSB1C3-K880005
· Transformation of comX and comW ligation product
· Digestion of comX-myc at EcoRI-HF and SpeI-HF
· Digestion of pCEPcin2 at EcoRI and EcoRV, XhoI and NcoI
· Ligation of 67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp P_combox with pSB1C3 backbone
· Ligate with E0240 PcomX
· Colony PCR for comE mutant
· Check sequence PcomCDE
·

Week 3

Aug 11
Wet lab
· Digestion check of comX-myc and comW-FLAG on XbaI and PstI-HF
· Gel purification of comX-myc digested on EcoRI-HF and SpeI
· Ligation of comX-myc cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI
· Restriction check of pSB1C3-P_combox (100bp, 180bp and 249bp) at XbaI and PstI
· Colony PCR of pSB1C3-P_combox (67bp, 100bp, 150bp, 160bp, 180bp, 249bp and 300bp)
· Gel purification of digested pSB1C3 backbone
· Digestion of pSB1C3-BBa_J04450 at XbaI and PstI
· Extract pSB1C3 from J04450
· Redo ligation part 1
· Gibson assembly of comE^D58E
· Ligation of PCR product comD to pSB1C3

Aug 12
Wet lab
· Digestion check of pSB1C3-K880005.comX and pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF
· Dilution of comW-FLAG
· Re-digestion of BBa_B0015 on EcoRI-HF and XbaI, digestion clean-up
· Re-ligation of pSB1C3-B0015.comX-myc
· Transformation of pSB1C3-B0015.comX-myc ligation product
· Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX-myc
· Digestion of pCEPcin2 at EcoRI and EcoRV
· Restriction check of pSB1C3-P_combox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI
· Ligate K880005 and comE(D58E)
· Inoculate for glycerol stock (PcomX)
·

Aug 13
Wet lab
· Digestion check of pSB1C3-B0015.comX-myc and pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Inoculation of pSB1C3-P_combox (100bp, 150bp, 160bp, 180bp and 249bp)
· Restriction check of pSB1C3-P_combox (100bp, 150bp, 160bp, 180bp and 249bp) at XmnI, NcoI and SpeI
· Colony PCR of pSB1C3-P_combox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)
· Check new dNTP
· Restrict check of ligation product part 1
· Colony PCR for checking part 2 ligation product and gibson assembly
· Inoculation of PcomX for glycerol stock
·
·

Aug 14
Wet lab
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Transformation of ligated product pSB1C3-K880005.comX-myc.B0015
· Dilution of comW-FLAG
· Digestion of comW-FLAG on EcoRI-HF and SpeI-HF
· Ligation of comW-FLAG into pSB1C3-B0015 backbone
· Re-digestion of comW-FLAG on EcoRI-HF and SpeI-HF
· Gel check of the digested pSB1C3-P_combox (100bp, 150bp, 160bp, 180bp and 249bp) obtained on July 13
· Colony PCR of Colony PCR of pSB1C3-P_combox (67bp, 100bp, 150bp, 160bp, 180bp and 249bp)
· glycerol stock for PcomX
· colony PCR ligation product part 1
· colony PCR gibson assembly
· Run gel for PCR products
· Inoculation of comE^D58E
·

Aug 15
Wet lab
· Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up
· Colony PCR of comW-FLAG using Taq DNA Polymerase
· Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF
· Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Overnight ligation of BBa_B0015 into pSB1C3-K880005.comX-myc backbone at 16°C
· Transformation of pSB1C3-K880005.comX-myc.B0015 ligation product
· Ligation of comX-myc into pSB1C3-B0015 backbone
· Inoculation of pSB1C3-K880005.comW-FLAG and pSB1C3-K880005.comX-myc.B0015
· Preparation for submittion of samples to be sequenced
· Inoculation of pSB1C3-P_combox (100bp, 150bp, 160bp, 180bp and 249bp)
· colony PCR for ligation product part 1
· colony PCR for ligation product part 2
· colony PCR for gibson assembly
· Run gel for PCR products
· comE-D58E plasmid extraction
·

Week 4

Aug 18
Wet lab
· Transformation of ligated pSB1C3-K880005.comX-myc
· Ligation of comW-FLAG into pSB1C3-K880005 backbone, comX-myc into pSB1C3-K880005 backbone, BBa_B0015 into pSB1C3-K880005.comX-myc backbone
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF
· Digestion check of pSB1C3-K880005.comW-FLAG on SpeI-HF and PstI-HF
· Digestion of pSB1C3-K880005.comX-myc on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Inoculation of pSB1C3-K880005.comX-myc and pSB1C3-B0015
· Digestion of pSB1C3-K880005.comX-myc on SpeI-HF and PstI-HF, digestion clean-up
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Digestion of pSB1C3-K880005 on SpeI-HF and PstI-HF, digestion clean-up
· Sent out pSB1C3-P_combox (100bp, 150bp and 180bp) samples for commercial sequencing
· Digestion of pSB1C3-P_combox (100bp, 150bp, 160bp, 180bp and 249bp) at SpeI and PstI
· Gel purification of digested pSB1C3-P_combox (150bp, 160bp, 180bp and 249bp)
· Inoculation of pSB1C3-BBa_E0240
· Gel extraction and purification of E0240
· Bacterial inoculation of PcomX (PcomCDE(3))
· Gel extraction and purification of comE mutant
·

Aug 19
Wet lab
· Digestion of pSB1C3-B0015 on EcoRI-HF and XbaI, digestion clean-up
· Digestion check of comX-myc on XbaI and PstI-HF
· Ligation of pSB1C3-K880005.comX-myc cut on EcoRI-HF and SpeI-HF iinto pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
· Inoculation of pSB1C3-K880005.comW-FLAG and pSB1C3-K880005.comX-myc.B0015
· Gel purification of digested pSB1C3-P_combox (100bp)
· Ligation of pSB1C3-P_combox (100bp, 150bp, 160bp, 180bp and 249bp) with BBa_E0240 insert
· Transformation of pSB1C3-P_combox-BBa_E0240 (160bp and 249bp)
· Digestion of pSB1C3-BBa_E0240 at XbaI and PstI
· Gel purification of E0240 insert
· Digest PcomX(PcomCDE(3))-pSB1C3
· Make aliquotes for oligo - PcomCDE
· PcomCDE Oligo annealing
· Ligation of comE mutant insert with K880005
· Ligation of comE insert with K880005
· Gel electrophoresis of Gibson assembly mastermix checking
· Plasmid extraction of J04450(CHL), PcomX(PcomCDE(3))
· Bacterial inoculation of comE WT, J04450 (CHL)
·

Aug 20
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on SphI and SpeI-HF
· Digestion check of pSB1C3-K880005.comX-myc on XbaI and PstI
· Inoculation of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comW-FLAG
· Gel purification of digested pSB1C3-BBa_E0240
· Digestion of pSB1C3-180bp P_combox at SpeI and PstI
· Digestion clean-up of pSB1C3-180bp P_combox
· Ligation of pSB1C3-180bp P_combox with BBa_E0240 insert
· Transformation of pSB1C3-P_combox-BBa_E0240 (100bp, 150bp and 180bp)
· Colony PCR for ligated pSB1C3-P_combox-BBa_E0240 (160bp and 249bp)
· Inoculation of pSB1C3-P_combox-BBa_E0240 (160bp and 249bp)
· Ligation of E0240 insert with PcomX
· Transformation [E0240-PcomCDE exp+ -ve, pSB1C3-PcomCDE exp+ -ve]
· Ligation of comE^D58E insert with K880005
· Gel extract comE WT
· PCR check for comE^D58E-myc
· Digest J04450(CHL) Gel extraction
· Plasmid extraction of comE WT, J04450 (CHL)
·

Aug 21
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF
· Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI
· Colony PCR of pSB1C3-B0015 and reverse comX-myc using Taq DNA Polymerase
· Digestion of comW-FLAG on NotI-HF
· Digestion of comW-FLAG on EcoRI-HF and SpeI-HF
· Inoculation of pSB1C3-K880005.comW-FLAG
· Transformation pSB1C3-K880005.comW-FLAG ligation product and pSB1C3-K880005.comX-myc.B0015
· Restriction check of pSB1C3-P_combox-BBa_E0240 (160bp and 249bp) at NcoI
· Colony PCR for ligated pSB1C3-P_combox-BBa_E0240 (100bp, 150bp and 180bp)
· Inoculation of pSB1C3-P_combox-BBa_E0240 (100bp, 150bp and 180bp)
· Received sequencing result of pSB1C3-P_combox (100bp, 150bp and 180bp)
· Ligate PcomX with E0240 insert
· PCR, Restrict check Part I (PcomCDE)
· O/N Ligation w/ PcomCDE
· Extract comE WT-pSB1C3
· PCR and restrict check comE^D58E-myc
· Glycerol stock for PcomX
·

Aug 22
Wet lab
· Digestion check of comW-FLAG on EcoRI-HF and SpeI-HF
· Digestion of pSB1C3-K880005.comX-myc.B0015 on EcoRI-HF and SpeI-HF
· Inoculation pSB1C3-K880005.comX-myc.B0015
· Restriction check of pSB1C3-P_combox-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI
· PCR of pSB1C3-P_combox-BBa_E0240 (160bp and 249bp) using Vent polymerase
· Sent out pSB1C3-P_combox-BBa_E0240 (160bp and 249bp) for commercial sequencing
· Transformation of newly ligated (E0240)
· Bacterial inoculation of checked colony (E0240)
· O/N ligation w/ pSB1C3
· Ligation comE WT-pSB1C3
· Ligate comE^D58E-myc w/ K880005
·

Week 5

Aug 25
Wet lab
· Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI-HF into pSB1C3-B0015 backbone cut on EcoRI-HF and XbaI Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI-HF Transformation of pSB1C3-K880005.comW-FLAG Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, gel extraction and gel purification Inoculation of pSB1C3-K880005.comX-myc.B0015 for glycerol stock
· Received sequencing result of pSB1C3-P_combox (160bp and 249bp)
· Restriction check of pSB1C3-P_combox-BBa_E0240 (100bp, 150bp and 180bp) at NcoI and XmnI
· Digestion of pSB1C3-P_combox (100bp, 150bp and 160bp, 180bp and 249bp) at XbaI and PstI
· Gel purification of digested P_combox-BBa_E0240 (100bp, 150bp and 180bp) insert
· Ligation of PcomX-E0240
· Digestion of PcomCDE-E0240
· Colony PCR pSB1C3-PcomCDE, K880005-comE WT, K880005-comE^D58E-myc
·

Aug 26
Wet lab
· Digestion of pSB1C3-K880005.comX-myc.B0015 on SpeI-HF and PstI-HF, digestion clean-up
· Ligation of Combox + GFP cut on XbaI and PstI-HF into pSB1C3-K880005.comX-myc.B0015 backbone cut on SpeI-HF and PstI-HF
· Digestion of BBa_E0240 100 bp on XbaI and PstI-HF, gel extraction and gel purification
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
· Transformation of pSB1C3-K880005.comW-FLAG
· Ligation of comX, 100bp P_combox-BBa_E0240 and pSB3K3 backbone
· Gel pSB1C3-PcomCDE
· PcomX-E0240 Colony PCR check
· PcomCDE glycerol stock
· Restrict check K880005-comE WT
· Ligation w/ comE WT
· Restrict check K880005-comE^D58E-myc
·

Aug 27
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF, gel extraction and gel purification
· Sequencing of pSB1C3-K880005.comW-FLAG.B0015
· Ligation of pSB1C3-K880005.comW-FLAG into pSB1C3-B0015 backbone and Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone
· Transformation of pSB1C3-K880005.comW-FLAG
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015 and pSB1C3-K880005.comW-FLAG
· Colony PCR of pSB1C3-P_combox-BBa_E0240 (180bp and 249bp)
· Ligation of comX, 100bp P_combox-BBa_E0240 and pSB3K3 backbone
· Inoculation of pSB1C3-P_combox-BBa_E0240 (100bp, 180bp and 249bp), pSB1C3-P_combox (160bp)
· pSB1C3-PcomCDE
· PcomCDE sequencing
· PCR check K880005-comE WT
· PCR check K880005-comE^D58E-myc
· Ligate K880005-comE-myc
·

Aug 28
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF, gel extraction and gel purification
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Ligation of Combox + GFP into pSB1C3-K880005.comX-myc.B0015 backbone
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015 and pSB1C3-K880005.comW-FLAG
· Ligation of pSB1C3-K880005.comW-FLAG into pSB1C3-B0015 backbone
· Restriction check of pSB1C3-P_combox-BBa_E0240 (180bp and 249bp)
· Colony PCR of pSB3K3-comX-100bp P_combox-BBa_E0240
· pSB1C3-PcomCDE overnight ligation
· PcomX-E0240 ligation
· Gel extract I20260 and E0240
· Glycerol stock for K880005-comE WT
· Colony PCR for K880005-comE^D58E-myc
· Ligate K880005-comE-myc
· PCR comE^D58E-myc
· Ligation of Part 1 and Part 2
·

Aug 29
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF
· Digestion check of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI-HF
· Sent out pSB1C3-160bp P_combox for commercial sequencing
· Digestion of pSB1C3-160bp P_combox at SpeI and PstI
· Colony PCR for pSB3K3-comX-100bp P_combox-BBa_E0240
· Ligation of pSB1C3-160bp P_combox with BBa_E0240 insert
· Transform pSB1C3-PcomCDE
· Transform PcomX-E0240
· Ligate I20260 and E0240
· Ligate K880005-comE WT
· Ligate K880005-comE-myc
· Ligate K880005-comE^D58E-myc
· Gel check comE^D58E-myc
·

September 2014

Week 1

Sept 1
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI-HF
· Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on XmnI
· Inoculation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP
· Received the sequencing result of pSB1C3-160bp P_combox
· Colony PCR: pSB1C3-PcomCDE, PcomX-E0240, I20260+Part2, E0240+Part2, E0240+Part2, K880005-comE WT, K990005-comE-myc, K990005-comE^D58E-myc
·

Sept 2
Wet lab
· Ligation of pSB1C3-K880005.comX-myc.B0015.Combox.GFP cut on EcoRI-HF and PstI-HF into pSB3K3 cut on EcoRI-HF and PstI-HF
· Re-digestion of pSB1C3-K880005.comX-myc.B0015.Combox.GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification
· Restriction check of pSB1C3-160bp P_combox-BBa_E0240 at XmnI and NcoI
· Whole construct gel check
· Restrict check PcomX-E0240
· Restrict check Part 1 + Part 2
· Restrict check I20260 and E0240
· Restrict check K880005-comE WT
· Restrict check K880005-comE-myc
· Restrict check K880005-comE^D58E-myc
·

Sept 3
Wet lab
· Streak plate for pSB1C3-K880005.comX-myc.B0015.Combox.GFP
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
· Restriction check of pSB1C3-160bp P_combox-BBa_E0240
· Restrict check I20260-Part 2
· Restrict check E0240-Part 2
· Restrict check Whole construct
· Restrict check -ve of whole construct ligation
· Restrict check pSB1C3-PcomCDE
·

Sept 4
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG on XbaI and PstI-HF, gel extraction and gel purification
· Restriction of Combox + GFP on EcoRI-HF and PstI-HF, gel extraction and gel purification
· Ligation of Combox + GFP into pSB3K3 backbone cut on EcoRI-HF and PstI-HF
· Transformation of pSB3K3-Combox.GFP ligation product
· Digestion of pSB1C3-160bp P_combox-BBa_E0240
· Ligation of 160bp P_combox-BBa_E0240,comX and pSB3K3
· Restrict check I20260-Part 2, E0240-Part 2, I20260-Part 2, whole construct, -ve of whole construct ligation
· Restrict check PcomCDE-E0240
· Restrict check I20260, E0240, Whole construct 3,4,5
· Restrict check and colony PCR for pSB1C3-PcomCDE
· Restrict check K880005+comE^D58E-N-myc
· Restrict check K880005+comE-C-myc
·

Sept 5
Wet lab
· Inoculation of pSB3K3-Combox.GFP
· Sequencing for pSB1C3-PcomCDE
·
·

Week 2

Sept 8
Wet lab
· Digestion check of pSB3K3-Combox.GFP on XmnI
· Restrict check and plasmid PCR for PcomCDE-E0240
· Obtain sequencing results for PcomCDE-pSB1C3, whole construct (PcomX)
· K880005-comE/comE^D58E-myc digest
· Ligation of Part 1 and 2 (also I20260, E0240)
· Transformation
·

Sept 9
Wet lab
·

Sept 10
Wet lab
· Streak plate for pSB3K3-Combox.GFP
· Restrict check PcomCDE-E0240
· Obtain sequencing results for PcomCDE-pSB1C3
· Whole construct (PcomX)
· K880005-comE/comE^D58E-myc send to sequencing
· Ligation and transformation of Part 1 and 2 (also I20260, E0240)
·

Sept 11
Wet lab
· Restriction check of pSB3K3-comX-160bp P_combox-BBa_E0240 at EcoRI and PstI
· Obtain sequencing results PcomCDE-pSB1C3
· Gel electrophoresis of colony PCR product from 10/9 PcomCDE-E0240
· Bacterial inoculation of checked products (for sequencing) and streak plates
· Check colony fluorescene for whole construct (PcomX)
· Gel extraction and purification of E0240 and I20260
· Gel extraction and purification of PcomCDE-E0240
· Streak plate for K880005-comE-C-myc
·

Sept 12
Wet lab
· Restriction check of pSB3K3-160bp P_combox-BBa_E0240 at EcoRI and PstI
· Transformation of pSB3K3-160bp P_combox-BBa_E0240
· Make glycerol stock for PcomCDE-pSB1C3
· Gel electrophoresis of colony PCR product of PcomCDE-E0240 from 10/9; Bacterial inoculation of checked products (for sequencing); Streak plate
· Check colony and fluorescence for whole construct (PcomX)
· Streak plate for K880005-comE-C-myc
·

Week 3

Sept 15
Wet lab
· Inoculation of pSB1C3-K880005.comW-FLAG
· PCR check and inoculation of colonies of I20260/E0240-K880005-comE-C-myc, I20260/E0240-K880005-comE^D58E-N-myc
· Restrict check, Gel extraction and Gel purification of pSB3K3 J04450 (CHL)
·

Sept 16
Wet lab
· Digestion of pSB1C3-K880005.comW-FLAG on EcoRI-HF and SpeI
·

Sept 17
Wet lab
· Digestion of pSB1C3-K880005.comW-FLAG on XbaI and PstI
· Ligation of pSB1C3-K880005.comW-FLAG cut on EcoRI-HF and SpeI into pSB1C3-B0015 cut on EcoRI-HF and XbaI
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI
· Digestion check of pSB1C3-K880005.comX-myc.B0015.Combox on XmnI
· Digestion check of Combox + GFP on XmnI and PstI
· Restriction check of pSB3K3-160bp P_combox-BBa_E0240 at NotI and PstI
· PCR check and inoculation for I20260/E0240-K880005-comE-C-myc, I20260/E0240-K880005-comE^D58E-N-myc
· Restrict check pSB3K3
· Gel extraction and Gel purification of J04450 (CHL) insert X,S
· Sequencing reaction for K880005-comE^D58E-N-myc, K880005-comE-C-myc
·

Sept 18
Wet lab
· Ligation of comX-myc into pSB1C3 cut on XbaI and PstI
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
· Colony PCR and inoculation of correct colonies for I20260/E0240-K880005-comE-C-myc, I20260/E0240-K880005-comE^D58E-N-myc
· Gel extraction and gel purification for J04450 (CHL), J04450 (KAN)
· Sequencing reaction and Restrict check for K880005-comE^D58E-N-myc, K880005-comE^D58E-N-myc
·

Sept 19
Wet lab
· Digestion check of pSB1C3-K880005.comW-FLAG.B0015 on XbaI and PstI
· Colony PCR, inoculation for I20260/E0240-K880005-comE^D58E-N-myc
· Miniprep for I20260/E0240-K880005-comE-C-myc
· Gel extraction and gel purification J04450 (CHL), J04450 (KAN) XS
· Sequencing reaction for K880005-comE-C-myc
·

Week 4

Sept 22
Wet lab
· Digestion check of pSB1C3-K880005.comX-myc.B0015 on XbaI and PstI
· Inoculation of pSB1C3-K880005.comW-FLAG.B0015
· Ligation of pSB3K3 backbone with 160bp P_combox-BBa_E0240
· Gel extraction and gel purification of I20260/E0240-K880005-comE^D58E-N-myc EP
· Colony PCR and inoculation of I20260/E0240-K880005-comE-C-myc
· Gel extraction and gel purification J04450 (CHL), J04450 (KAN)
· Oligo annealing and overnight ligation w/ pSB1C3for PcomCDE
·

Sept 23
Wet lab
· Digestion of pSB1C3-K880005.comW-FLAG.B0015 of EcoRI-HF and SpeI-HF
· Digestion of pSB1C3-K880005.comX-myc.B0015 and pSB1C3-K880005.comX on EcoRI-HF and SpeI-HF, gel extraction and gel purification
· Ligation of pSB1C3-K880005.comX into pSB1C3-B0015 cut on EcoRI-HF and XbaI
· Transformation of pSB3K3-160bp P_combox-BBa_E0240 ligated on Sept 22
· Gel check and redo liagtion for I20260-K880005-comE^D58E-N-myc, E0240-K880005-comE^D58E-N-myc, I20260-K880005-comE-C-myc, E0240-K880005-comE-C-myc
· Gel extraction and purification for I20260-K880005-comE^D58E-N-myc, E0240-K880005-comE^D58E-N-myc
· Ethanol precipitation for sequencing, restrict check K880005-comE^D58E-N-myc, K880005-comE-C-myc
· Dephosphorylation and gel extraction and gel purification of J04450 (CHL) (X&S)
· Gel extraction and Gel purification, dephosphorylation of J04450 (KAN) (E&P)
· Oligo annealing, overnight ligation w/ pSB1C3 of PcomCDE
·

Sept 24
Wet lab
· Digestion check of pSB1C3-K880005.comX.B0015 on XbaI and PstI, gel extraction and gel purification
· Inoculation of bacterial colonies carrying pSB3K3-160bp P_combox-BBa_E0240
· Restrict check, gel extraction and purification, Ligation w/ pSB3K3 I20260-K880005-comE^D58E-N-myc, E0240-K880005-comE^D58E-N-myc, I20260-K880005-comE-C-· myc, E0240-K880005-comE-C-myc
· Obtain sequencing results for K880005-comE^D58E-N-myc, K880005-comE-C-myc
· Transform pSB1C3-PcomCDE
·

Sept 25
Wet lab
· Restrcition digestion of ligated pSB3K3-160P_combox-BBa_E0240
· Restrict check, gel extraction and purification, Ligation w/ pSB3K3, I20260-K880005-comE^D58E-N-myc, E0240-K880005-comE^D58E-N-myc, I20260-K880005-comE-C-myc, E0240-K880005-comE-C-myc
· Obtain sequencing results for K880005-comE^D58E-N-myc, K880005-comE-C-myc
· Colony PCR w/ ‘NEW FPcomCDE’ and VR, inoculation of pSB1C3-PcomCDE
·

Sept 26
Wet lab
· Transformation of pSB3K3-160P_combox-BBa_E0240 sample1
· Streak plate for pSB3K3-BBa_I20260 and pSB3K3-BBa_E0240
· Gel extraction and purificationI and ligation20260-K880005-comE^D58E-N-myc, E0240-K880005-comE^D58E-N-myc, I20260-K880005-comE-C-myc, E0240-K880005-comE-C-myc
· Restrict check K880005-comE^D58E-N-myc, K880005-comE-C-myc
· Inoculation for sequencing K880005-comE^D58E-N-myc, K880005-comE-C-myc
· Transformation of pSB1C3-PcomCDE
·

Week 5

Sept 29
Wet lab
·

Sept 30
Wet lab
· Digestion of RBS+comX-myc+B0015 on XbaI and PstI
· Ligation of PcomCDE-E0240, both that day and overnight
· Transformation
·

Riboregulator

July 2014

Week 1

July 7
Wet lab
. Oligos phosphorylation
. ligation of TAs and CRs
. Colony PCR
. Digest Receptor Backbone pSB1C3
. Agar-LB plates
. Inoculation for transformed Biobricks
. Primer Design for TA construct Gibson Assembly
. Gel check for the colony PCR
. P_bad PCR amplification

July 8
Wet lab
. Transformation of ligated TAs and CRs
. Miniprep previous TAs CRs constructs
. restriction check previous TAs CRs constructs
. Colony PCR for CRs and TAs
. Re-ligation of TAs and CRs
. P_bad PCR clean up and Digestion
. Ligation of P_bad with pSB1C3
. Sequencing preparation of P_bad

July 9
Wet lab
. Transformation of Re-ligated TAs and CRs
. Colony PCR
. Gibson assembly master mix preparation

July 10
Wet lab
. Sequencing preparation of P_bad
. Miniprep previous TAs CRs constructs
. restriction check previous TAs CRs constructs
. Colony PCR
. Inoculation for glycerol stocks
. Digest Receptor Backbone , digestion of BBa_ BBa_E0240 with EcoR1-HF and Xbal and gel purification

July 11
Wet lab
. P_bad digestion and gel purification, Digestion of P_bad-pSB1C3 with EcoR1-HF and Spe1 and gel purification
. Ligation of P_bad and BBa_E0240
. Overlapping PCR design
. Gibson assembly master mix preparation
. Glycerol stock
. Competent cells preparations for DH5alpha and DH10B
. Run gel for yesterday's restriction check
. Miniprep BBa_E0240, mRFP, BBa_B0015
. Colony PCR
. One full set re-ligation (short time)
. Miniprep BBa_I13401 and BBa_K592009

Week 3

July 14
Wet lab
. Gibson assembly master mix preparation
. Trevor's PCR
. Ligation of P_bad an BBa_E0240
. Competent cells preparations for DH5alpha and DH10B
. Prepare receptor backbone for oligo anealing, pSB1C3-BBa_J04450 (EcoRI, SpeI)
. One full set of re-ligation (short time)
. One full set of re-ligation (O/N)
. Preparations for P_bad re-Characterization

July 15
Wet lab
. Inoculation for P_bad -GFP-TT
. (2nd time) Colony PCR using VF2 and VR
. Design primer for P_bad promoter sequencing
. Gibson assembly master mix preparation
. Preparations for P_bad re-characterization
. Competent cells preparations for DH5alpha and DH10B
. One full set re-ligation (O/N)
. Prepare receptor backbone for oligo anealing

July 16
Wet lab
. Gibson assembly master mix preparation
. Preparations for P_bad re-characterization
. Competent cells preparations for DH5alpha and DH10B
. Prepare receptor backbone for oligo anealing
. inoculate successful oligo annealing colonies
. Oligo annealing products C. PCR?

July 17
Wet lab
. Gibson assembly master mix preparation
. P_bad re-characterization
. Miniprep CRs TAs
. Restriction check CRs,TAs
. Oligo annealing products C. PCR
. Prepare receptor backbone for oligo anealing
. Restrict CRs plasmids for GFP.dTT
. Restrict TAs plasmids for dTT
. Restrict GFP.dTT for CRs
. Restrict dTT for TAs
. Ligate CR.GFP.dTT
. Ligate TA.dTT
. Transform Ligated CR.GFP.dTT
. Transform Ligated TA.dTT
. Transform-Ligate Annealed oligos

July 18
Wet lab
. Gibson assembly for amilCP
. P_bad re-characterization
. Colony PCR
. Prepare receptor backbone for oligo anealing
. Restrict GFP.dTT for CRs
. Restrict dTT for TAs
. M9 mininaml medium preparation
. Colony PCR
. plate streaking for P_bad-BBa_E0240
. inoculation of TAs and CRs

July 19
Wet lab
. P_bad re-characterization

Week 4

July 21
Wet lab
. Gibson assembly for amilCP
. Miniprep CRs TAs
. restriction check pSB1C3-TAs/CRs constructs
. Restrict GFP.dTT for CRs
. Restrict P_bad for TAs
. Restrict BBa_B0015 EcoR1 and XbaI
. BBa_J04450 backbone extraction
. Colony PCR
. inoculation of TAs and CRs
. prepare P_badL(+) A. concentration gradients for characterization
. Restrict CRs
. Restrict TAs-Version1
. Restrict TAs-Version2
. Ligate CR.GFP.dTT
. Ligate TA.dTT
. Transform P_bad.TA
. Transform CR.GFP.dTT
. Transform Gibson assembly

July 22
Wet lab
. Colony PCR
. Miniprep CRs TAs
. restriction check pSB1C3-TAs/CRs constructs
. Restrict BBa_B0015-Version2

July 23
Wet lab
. Inoculation candidate colony or construct
. Miniprep and restriction check inoculations
. Gibson assembly check
. Gibson PCR
. Teach inoue competent cells to main project
. Miniprep of BBa_B0015, P_bad-BBa_E0240
. glycerol stock (P_bad-BBa_E0240)
. Re-ligation of oligo annealing O/N

July 24
Wet lab
. miniprep candidate colony or construct
. restriction check pSB1C3-TAs/CRs constructs
. Re-Re-ligation transformation
. Re-Re-ligation transformation V.X
. Gibson assembly check

July 25
Wet lab
. Certification day (safety evaluation)

Week 5

July 28
Wet lab
. oligo phosphorylation
. oligo annealing
. ligation of annealed oligos O/N
. gel for C. PCR
. receptor backbone for annealed oligos
. inoculate last ligation
. Check past locks/keys status
. P_bad sequencing
. transformation of some of the existing riboregulator Biobricks
. Benchling Check

July 29
Wet lab
. Miniprep and restriction check inoculations
. restriction check pSB1C3-TAs/CRs constructs
. Inoculation of CR and TA from distribution kit
. prepare BBa_I20260 in pSB1C3
. prepare Side project Buffer
. prepare side project gel red 200:1
. Check digested GFP
. Check digested BBa_B0015
. ligate oligos into GFP
. ligate oligos into BBa_B0015
. prepare M9 for characterization
. transform our P_bad into DH5alpha
. prepare M9 medium
. transformation of Clon001-010 in pSB1C3 ( with a control)

July 30
Wet lab
. transformation of CLON001-010 in pSB1C3 (BBa_B0015, BBa_I13401)
. C. PCR for yesterday transformations (CLON001-010)
. If construct after gel check inoculation, restriction, ligation
. prepare BBa_I20260 in pSB1C3
. transform our P_badinto DH5alpha
. prepare M9 for characterization
. Teach inoue competent cells to main project

July 31
Wet lab
. Try Gibson assembly mastermix
. Digest Biobrick TA and CR
. PCR clean up? for existing CR and TA Biobricks
. run gel for yesterday's colony PCR
. check dNTPs
. Sequencing preparation for possible construct
. colony PCR for 5th
. inoculate candidates
. prepare BBa_I20260 in pSB1C3

August 2014

Week 1

Aug 1
Wet lab
. colony PCR for 5th
. Digest Biobrick TA and CR
. short ligation for TAs CRs
. Transform TAs, CRs
. inoue Buffer
. inoculations if possible
. miniprep CLON005
. Digest 005 Spe1 and Pst1
. ligate CLON 005 with BBa_B0015
. prepare backbone : BBa_B0015 E, X /// BBa_I13401 E,X /// J23102 X, P
. Ligate P_bad with Key1 dTT
. digest P_tet lock 1 with S and P

Aug 2
Wet lab
. Devise a general blue print for the catalog and feature page

Week 2

Aug 4
Wet lab
. Mini prep 001-010
. Restrict TAs
. restrict CRs
. Restrict P_bad for TAs
. Restrict GFP.dTT for CRs
. Restrict BBa_B0015
. Ligate P_bad.TA
. ligate CR.GFP.dTT
. Ligate P_bad.dTT
. Transform TAs, CRs
. inoculate 001-010
. Sequence 001-010
. inoculate BBa_B0015
. inoculate constitutive promoter
. inoculate P_bad with key 1 dTT
. Competent cells preparations DH10B
. Prepare for functional assay of P_bad promoter
. Prepare M9 minimal Medium
. make a second list of oligos to be sent out
. Test out GA mastermix efficiency
. Ligate TAs oligo with BBa_B0015
. inoue Buffer
. Sequence 001-010

Aug 5
Wet lab
. Colony PCR for TA.dTTs
. inoculate TA.dTT
. ligation of P_bad and key3
. Restrict TAs
. restrict CRs
. Restrict P_bad for TAs
. Restrict GFP.dTT for CRs
. Ligate P_bad.TA
. ligate CR.GFP.dTT
. Ligate P_bad.dTT
. Transform TAs, CRs
. Prepare M9 medium
. Prepare inoculations for P_bad characterization
. Sequecing preparation
. Digest P_tet lock 1 with S and P
. Glycerol stock for 001-010
. Inoue competent cells
. "Mini Prep pSB1C3-PCRP_bad

Aug 6
Wet lab
. Miniprep TA.dTT
. Restriction check for TA.dTT
. Miniprep TAs CRs
. Restrict TAs
. Restrict CRs
. Restrict BBa_B0015 for TAs
. Colony PCR for TA.dTTs
. Oligo annealing for TA.dTT
. Inoue competent cells
. Sequencing results
. Ligate P_bad with Key1 dTT
. Ligate P_tet Lock 1 (GFP dTT)
. Send PCR P_bad for sequencing
. Assay P_bad promoter

Aug 7
Wet lab
. Gel check for C. PCR
. inoculate possible TA.dTT
. Ligation of TA and dTT (4TAs)
. Ligation of P_bad and key dTT
. Gel check for TAs CRs
. competent cells
. miniprep P_bad-dTT
. miniprep P_bad-key3
. Colony PCR for P_bad-dTT and P_bad-key3
. Restriction of P_bad-dTT and P_bad-key3
. prepare backbone and or insert if any // and restriction check
. Preparation for P_bad promoter characterization
. Colony PCR for P_bad with Key 1 dTT
. Colony PCR for P_tet Lock 1 GFP dTT
. inoculation of ligated DNA

Aug 8
Wet lab
. digestion of P_bad-key3
. Restriction check for P_bad-dTT and P_bad-key3-DTT
. ligation of P_bad-key3-dTT
. Miniprep for CLON007(J), CLON 010(I) and CLON (G)
. Miniprep P_bad Key 1 dTT
. Miniprep P_tet Lock 1 GFP dTT
. Restriction Check for the key 1 and lock 1 mentioned above
. Prepare digestion of key 1 and lock 1 for ligation
. Transform BBa_J13002
. Restriction check CLON007(J), CLON 010(I) and CLON (G)
. digestion of CLON007(J), CLON 010(I) and CLON (G)
. ligation of CLON007(J), CLON 010(I) and CLON (G) with P_bad
. C. PCR for CLON 022-025 TAs.dTT
. ligation of P_bad and TAs
. ligation of GFP and CRs
. Inoue competent cells
. P_bad promoter characterization

Aug 9
Wet lab
. transformation , all in CHL, overnight ligation 16C
. Colony PCR gel check
. Check competent cells

Aug 10
Wet lab
. Restriction check for TAs.dTT
. Gel check for C. PCR TAs.dTT

Week 3

Aug 11
Wet lab
. Restriction check for TAs.dTT
. Gel check for C. PCR TAs.dTT
. Colony PCR for CR.GFP.dTTs
. Colony PCR for P_bad.TAs
. Inoculate CR.GFP.dTTs
. Inoculate P_bad.TAs
. Restrict TA.dTTs
. Ligate P_bad.TA.dTTs
. Transform P_bad.TA.dTTs
. Restriction check for P_tet Lock 1 GFP dTT and P_bad Key 1 dTT
. Transform 2014 Kit plate 4 well GF
. Transformation of restricted checked DNA
. inoculate BBa_J13002
. inoculate more P_bad dTTs
. sequencing PCR for MCR, MTA
. transformation for backbone with KAN resistant
. inoculation B0034 from glycerol stock B0034
. restriction check
. Ligation of P_bad-key3 into BBa_B0015-pSB1AK3 plasmid
. inoculation of P_bad-key3

Aug 12
Wet lab
. transformation P_bad -TA
. Gel for TA.dTTs
. GEl for C. PCR for CR.GFP.dTT
. Digestion of PCR P_bad

Aug 13
Wet lab
. miniprep CR.GFP.dTTs
. miniprep P_bad
. restriction check CR.GFP.dTTs
. inoculate TA.dTTs
. Gel extraction-purification of constitutive promoter
. Ligate CR.GFP.dTTs into constitutive promoter
. transform, P_bad.Key3
. transform constitutive promoter.CR.GFP.dTTs
. Gibson Assembly mastermix
. Digest P_bad Key dTT and P_tet Lock 1 GFP dTT for 3A assembly
. Miniprep pSB3K3-J04550 for pSB3K3 Backbone
. 3A assembly
. Plan for P_bad characterization
. Prepare stock arabinose solution M9 medium

Aug 14
Wet lab
. Gel check for lock3.GFP.dTT colony PCR I-VI
. check dNTPs
. miniprep TA.dTTs
. restriction check TA.dTTs
. miniprep Lock3.GFP.dTT
. colony PCR for P_bad-key3-dTT
. Gel extraction-purification of constitutive promoter
. Ligate CR.GFP.dTTs into constitutive promoter
. ligate TA.dTT with P_bad
. transform ligate P_bad.TA.dTTs
. transform constitutive promoter.CR.GFP.dTTs

Aug 15
Wet lab
. Gibson Assembly mastermix
. purify CR.GFP.dTTs
. Ligate CR.GFP.dTTs into constitutive promoter
. Digestion of TA-dTT and PCR clean-up
. Restriction Check for TA-dTT
. transform ligate P_bad.TA.dTTs
. transform constitutive promoter.CR.GFP.dTTs
. make 10x DNA loading Dye
. Order new TA and CR
. Gibson Assembly mastermix

Aug 16
Wet lab
. Miniprep
. Colony PCR for final constructs

Week 4

Aug 18
Wet lab
. Restriction check for P_bad.TA.dTTs
. Restriction check for constitutive promoter.CR.GFP.dTT
. Restriction check for constitutive promoter.RBS.GFP.dTT
. PCR for CR.GFP.dTT
. PCR for RBS.GFP.dTT
. PCR check for constitutive promoter.CR.GFP.dTT
. PCR check for constitutive promoter.RBS.GFP.dTT
. PCR check for P_bad.TA.dTT
. restriction check TA.dTTs
. Prepare functional assay for RBS, CRs
. Inoculate candidates
. Prepare pSB3K3
. Prepare pSB1C3 for oligo ligation
. Prepare BBa_I13401 for oligo ligation
. Prepare BBa_B0015 for oligo ligation
. Inoue competent cells
. Prepare for P_bad characterization
. Take over the tank B and C around 1pm-5;30

Aug 19
Wet lab
. Gel check for O/NPCR products
. Gel check for PCR products (18/08)
. miniprep c.Promoter.Lock 1RBS.GFP.dTT
. miniprep possible candidates
. Restrict TA.dTT
. pSB1C3-BBa_I13401 for oligo ligation
. BBa_B0015 for oligo ligation
. pSB1C3-BBa_J04450 for oligo ligation
. Inoue competent cells
. P_bad-key3-dTT restriction check

Aug 20
Wet lab
. Gel check for P_bad.TA.dTTs
. purification for BBa_B0015, BBa_I13401
. pSB1C3-BBa_J04450 for oligo ligation
. Gel check for PCR products (19/08)
. PCR check for J23102, LOCK 3 GFP DTT
. Restriction check for key1 DTT

Aug 21
Wet lab
. Colony PCR for final constructs
. Ligate UST lock
. restriction check for key1.dTT
. ligate P_bad.key1.dTT
. ligate lock 3.GFP.dTT with C. promoter
. Colony PCR for c. promoter.MCR.GFP.dTT
. competent cells
. Glycerol stocks
. transform HKUSTlock.GFP.dTT
. Transform pSB1C3-HKUSTlock

Aug 22
Wet lab
. competent cells
. Colony PCR for pSB1C3-HKUSTlock1
. Colony PCR for HKUSTlock1.GFP.dTT
. miniprep pSB3K3-J04550 for pSB3K3 Backbone
. restrict pSB3K3 for 3 pieces assemblage
. ligate TA-CR into pSB3K3
. Transform TA-CR
. colony PCR for P_bad-key3-dTT
. inoculate P_bad.key1.dTT
. gel check for C. PCR on 21/08
. extraction of pSB3K3
. inoculate HKUSTlock1.GFP.dTT
. inoculate pSB1C3-HKUSTlock1

Aug 23
Wet lab
. Gel check for TAs
. colony PCR for UST lock in BBa_I13401 and pSB1C3
. transform ust key
. Check competent cells

Aug 24
Wet lab
. Colony PCR for P_bad.Key1.dTT
. Gel check for P_bad.Key1.dTT

Week 5

Aug 25
Wet lab
. Colony PCR for P_bad.Key1.dTT
. PCR check for CR.GFP.dTT
. PCR check for Cons. prom.CR.GFP.dTT
. anneal and ligate ust lock
. anneal and ligate ust key
. transform ust lock1
. transform ust key
. ligation again key 1 into BBa_B0015

Aug 26
Wet lab
. Colony PCR for "un-named lock"
. inoculate ones which SEEMS like it contains lock
. Colony PCR for "un-named key"
. inoculate ones which SEEMS like it contains key
. colony PCR for Key1.DTT
. Gel check for O/NPCR products
. PCR check for CR.GFP.dTTs
. Inoculate all possible candidates
. Feeling paranoic? Prepare BBa_I13401 for CRs
. Streak possible c.p.CR/RBS.GFP.dTT
. make SOC
. single cut check for TAs
. make 5 alpha cell

Aug 27
Wet lab
. miniprerp candidates
. PCR check for candidates
. gel check for o/n PCR
. restriction check for ust key 1
. restriction check for key 1
. P_bad for ust key 1
. extract ust lock 1 for j23102
. ligate ust lock and key
. transform ust lock and key
. prepare plates for macroscope
. prepare dna for sequencing
. SOB
. SOC
. oligo annealing
. plasmid PCR for HKUSt lock and key
. overnight digestion of B0034 for control construct
. Digestion of HKUST key-BBa_B0015

Aug 28
Wet lab
. miniprep locks
. glycerol stocks for locks
. minipreps for BBa_B0015 and B0034
. ligation of P_bad-Hkust key-BBa_B0015
. digestion of HKUST lock
. ligation of HKUST lock with GFP
. ligation of HKUST key and lock (4th)
. ligation of B0034 with GFP
. colony PCR of HKUST key and lock
. inoculation of key and lock
. miniprep
. Key one restriction check
. key one restriction for inserting P_bad
. Restrict and exytact lock3c rbs and ligate into c.promoter
. ligate lock 3c.GFP.dTT with C. promoter
. transform c. promoterlock 3c rbs.GFP.DTT
. Extract mcr/mta
. ligate MCR and MTA into pSB3K3
. transform MCR/MTA
. Glycerol stock for locks
. BBa_E0240 ligate with C. Promoter
. TB
. parallel ligation and aneling for key 1
. Sequencing preparations

Aug 29
Wet lab
. minipreps
. Gel extraction, purification for O/N digestions
. competent cells
. Extract mcr/mta
. ligate MCR and MTA into pSB3K3
. transform MCR/MTA
. Glycerol stock for locks
. PCR check for Biobrick key1

Aug 30
Wet lab
. inoculation of 12 falcons
. Colony PCR for pSB3K3 constructs
. Transform key1 O/N
. miniprep
. Competent cells
. AseI check Key 1 Dtt

September 2014

Week 1

Sept 1
Wet lab
. restriction check P_bad- Lost your key-DTT
. restriction check P_bad-Lost your key-DTT
. colony PCR for CR.TAs (orthogonality)
. colony PCR for Key1,DTT
. ligate lock3c.GFP.dTT into c.p plasmid
. transform lock3.GFP.DTT
. Colony PCR for CR.TAs (orthogonality)
. Colony PCR for sherLock-GFP-DTT
. restriction check for Lost Your Key in pSB1C3
. transform P_bad.lost your key.DTT
. inoculate orthogonality candidates
. Prepare midi-prep for pSB3K3

Sept 2
Wet lab
. Gel check for O/N colony PCR for key1 and pSB3K3
. massive prep
. PCR check sense reverse
. colony PCR for orthogonality
. colony PCR for cons. pro. lock3crbs.GFP.DTT
. migrate cpromoter BBa_E0240 to pSB3K3 with mta key
. inoculation of B0034-GFP-DTT
. inoculation of Sherlock-GFP-DTT
. sequencing for sherlock in pSB1C3
. digestion of B0034-GFP-DTT
. digestion of sherlock-GFP-DTT

Sept 3
Wet lab
. digestion of P_bad MTA dTT
. digestion of P_bad dTT
. digestion of J23102 MCR GFP dTT
. digestion of J23102 BBa_E0240(B0032 GFP dTT)
. digestion of pSB3K3 backbone (from pSB3K3-BBa_J04450)
. Ligation of J23102 MCR GFP dTT with P_bad dTT
. Ligation of J23102 BBa_E0240 with P_bad MTA dTT
. Ligation of J23102 BBa_E0240 with P_bad dTT
. Plasmid PCR for SherLock-GFP-DTT
. ligation of B0034-GFP-DTT
. ligation of SherLock-GFP-DTT
. sequencing
. check plate for arabinose induced mcr mta while singing
. extraction of key and lock
. restriction check for key with lock
. extraction of key1 DTT
. ligation of key1 DTT with P_bad
. Gel check for O/N colonyPCR
. CRAZY PCR check
. Check 031
. ligate lock3c RBS.GFP.dTT into c.p plasmid
. transform lock3c RBS.GFP.dTT
. Midi-prep preparations

Sept 4
Wet lab
. Gel purification of digested insert and backbone
. Ligation of J23102 MCR GFP dTT with P_bad dTT
. Ligation of J23102 BBa_E0240 with P_bad MTA dTT
. digestion of J23102 BBa_E0240(B0032 GFP dTT)
. Transformation of ligated projects mentioned above
. Colony PCR for sherLock-GFP-DTT
. Colony PCR for B0034-GFP-DTT
. Inoculation of successful colonies
. Gel check for O/N Colony PCR
. Gel check for O/N crazy PCR
. Gel check for Erics restriction check
. Inoculation of successful colonies
. Midi-prep preparations
. Gel purification

Sept 5
Wet lab
. miniprep/ glycerol stock
. Transform P_bad dTT
. Transform ligated produce of J23102 MCR GFP dTT with P_bad dTT

Week 2

Sept 8
Wet lab
·
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·
·
·
·
·

Sept 9
Wet lab
·
·
·
·
·
·

Sept 10
Wet lab
. colony PCR
. ligations for final constructs
. inoculations
. P_bad.lost your key.DTT
. sherlock
. gel check for colony PCR
. colony PCR

Sept 11
Wet lab
·
·
·
·

Sept 12
Wet lab
. colony PCR
. restrict key1.DTT for P_bad insertion
. ligate P_bad with key1.DTT
. transform key1.dTT
. extract pSB3K3
. Prepare TB
. Prepare dextrose for SOC
. check sequencing results
. Digestion of CLON041 for constructs
. ligation
. inoculation
. Digestion of CLON 036
. colony PCR mcr mta

Week 3

Sept 15
Wet lab
·
·
·
·

Sept 16
Wet lab
. mini prep P_bad.key1.DTT
. restriction check P_bad.key1.DTT
. gel check for O/N c.PCR
. extract P_bad.key1
. inoculate every CR.TA construct
. ligate transform key 1 with locks
. miniprep (B1, B2, and B3)
. plasmid PCR B1, B2 and B3 using sense oligo and VR
. Overnight digestion for ones which give positive results

Sept 17
Wet lab
. purify P_bad.key1
. ligate P_badkey1 DTT with locks
. transform locks.P_bad.key1.DTT
. 2xYT (agar) AMP
. transform all

Sept 18
Wet lab
. Spread a plate
. miniprep for 3 inoculations
. Sense oligo and VR PCR for HKUST Lock-DTT construct
. Overnight digestion of plasmid which gives a band around 1300bp
. EtoHpp for sequencing
. Miniprep constructs for submission
. inoculation
. transform 031, 034
. Check "Diagonal constructs" and controls
. migrate CR constructs into pSB3K3

Sept 19
Wet lab
. miniprep lock.key candidate
. ligate problematic cr.key
. restriction check sherlock
. extractions.... madness

Sept 20
Wet lab
. miniprep CR.TA
. Colony PCR for CR.TA
. Colony PCR for CR.TA orthogonality

Sept 21
Wet lab
. miniprep matching CR.TA
. Restriction check by EcoR1 and Pst1

Week 4

Sept 22
Wet lab
. Run Gel check for matching set of CR.TA
. ligate problematic sets
. transform problematic sets
. prepare DNA for submission
. Gel purification for Doyle
. Ligation lock-DTT with constitutive promoter
. transformation
. pSB3K3-CRs ligation

Sept 23
Wet lab
. pSB3K3-CRs transformation
. Gel extractions
. Gel purifications
. TB
. Agar L (+) plates
. Send DNA to BGI for sequencing
. C. PCR?
. Inoculate possible candidates
. ligate problematic sets
. transform confirmed constructs
. inoculation for HKUST lock Construct
. colony PCR For lock

Sept 24
Wet lab
. Competent cells preparations
. minipreps
. colony PCR
. miniprep for Biobrick submission
. send plasmid for sequencing

Sept 25
Wet lab
. insane prep
. restriction check
. Gel check for previous PvuII digestions

Sept 26
Wet lab
. Gel check PvuII digestions
. Gel check AseI digestions
. 3A assembly for non pSB1C3
. final batch of ligation for all missing component
. prepare DNA for submission
. Extraction for ligation
. Checking constructs-3 constructs using digestion check
. transformation for LOCK-8
. Restriction check for controls for MCR/MTA
. Transformation of confirmed plasmid

Sept 27
Wet lab
. C. PCR for H series...
. inoculate possible candidates
. check self ligated pSB3K3
. Gel check for possible candidates AseI
. Gel check for possible candidates PvuII
. Ligated H6 H7
. transformed H6 H7
. O/N inoculation of MCR/MTA for characterization

Sept 28
Wet lab
. Extract pSB1C3
. Extract 036
. miniprep "H" series candidates
. Repeat CHL restrictions
. Repeat CHL ligations
. Repeat CHL transformations
. Gel check for "7" series using NcoI
. restriction check AseI
. restriction check NcoI
. Fluorescence measurement of MCR/MTA for characterization.
. Pick a colony dilute and spread MCR/MTA (we don't want bacteria lawn)

Week 5

Sept 29
Wet lab
. restriction check AseI
. restriction check NcoI
. mini prep 6 + MCR/MTA (thanks from Doyle)
. Restriction check for MCR/MTA in pSB3K3.
. restrict 036
. purify 036
. ligate monkey with sherlock
. Gel check for NcoI and AseI
. Get pSB1C3 backbone EcoRI and PstI
. Fluorescence measurement of MCR/MTA for characterization (second trial)
. inoculate Lock and Keys for crude functional assay.

Sept 30
Wet lab
. miniprep 11 tubes in shaker c
. restriction check H" series with NcoI
. Restrict inoculated colonies wiht AseI
. Restrict inoculated colonies wiht NcoI
. Gel purifications
. ligation of H series and some from other series...
. transformation of 16 confirmed constructs

Oct 1
Wet lab
. digestion of key3,HKUSTkey?
. miniprep- final constructs

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