15/04/22

Primer Design

Primer Design iILS1-9

15/04/26

Miniprep

15/06/03

PCR

PCR Mix	PCR1	PCR2	PCR3	PCR4	PCR5	PCR6
0,5ul	BB1 3108	GFP1 3504	BB2 3109	GFP2 3504	BB3 111	GFP3 3504
1ul	ILS2	ILS4	ILS2	ILS7	ILS2	ILS9
1ul	ILS3	ILS5	ILS6	ILS5	ILS8	ILS5
31,5ul	H2O	H2O	H2O	H2O	H2O	H2O
10ul	Buffer	Buffer	Buffer	Buffer	Buffer	Buffer
4ul	dNTPs	dNTPs	dNTPs	dNTPs	dNTPs	dNTPs
2ul	GXL Polymerase	GXL Polymerase	GXL Polymerase	GXL Polymerase	GXL Polymerase	GXL Polymerase

Cycler Protocol (30x)	PCR1,3,5	PCR2,4,6
98°C	10s	10s
55°C	15s	15s
68°C	22s	10s
end cycle
10°C	store	store

PCR

Repeat PCR of BB1, BB2
Comments: unsatisfied yield for PCR1, PCR3 -> pcr of pcr (3', 4') and repeat PCR with original template (1',2')

Gel Extraction

Gel extraction of PCR2,4,6

15/06/04

PCR

PCR of BB111 (BB3)

15/06/08

Gel electrophoresis

PCR1'-4'

PCR

PCR of GFP (PCR2,4,6)

Gel electrophoresis

PCR2,4,6

15/06/09

PCR

Repeating PCR of PCR1, PCR3, PCR4

Gel Extraction

Gel of PCR1,3,4

ILS — **Figure 3:** PCR1, 3, 4
PCR1 correct (longer band is the one)
PCR3??

DpnI digest

Add 2ul of DpnI to the PCR, incubate for 30min @37°C

Gibson Assembly

Gibson1: BB1+GFP1= pILS1
Gibson3: BB3+GFP3= pILS3

	Gibson 1	Gibson 2
BB	1,4ul	1,5ul
GFP	3,23ul	2,6ul
H2OC	5,4ul	5,9ul
Gibson MM	10ul	10ul

Transformation electrocompetent cells

Transform 2ul Gibson mix of Gibson 1 and Gibson3 into NEB electrocompetent cells
Recover 1h, plate 100ul

15/06/11

CPEC

	CPEC 1	CPEC 3
BB	3,34ul	3,78ul
GFP	0,69ul	0,56ul
H2OC	5,97ul	5,65ul

Analytical digest

BB2	4ul
H2O	3ul
XbaI	1ul
SacI	1ul
Cutsmart	1ul

30min @37°C

Gel electrophoresis

Check the digest of BB2

ILS — **Figure 4:** Problem: Just one band visible; repeat with other enzymes and load undigested BB

15/06/12

Analytical digest of BB2

Digest 1		Digest 2		Digest 3
BB2	4ul	BB2	7ul	BB2	4ul
H2O	3ul			H2O	2ul
PvuII	1ul	EcoRI	1ul	EcoRI	1ul
PstI	1ul	PstI	1ul
Cutsmart	1ul	Cutsmart	1ul	Cutsmart	1ul

Incubate 30min @37°C

Gel Extraction

Check the different digests of BB2

ILS — **Figure 5:** undigested - digest 1 - digest 2 - digest 3
Something is wrong - they don'r show the correct size at all!!! Check registry and wells

15/06/13

Transformation chemocompetent cells

Transform following plasmids into NEB Turbo (Chemical competent):
K823005 Promoter
K823008 Promoter
K823013 Promoter
Gibson 1
Gibson3
CPEC1
CPEC3

15/06/15

Inoculation

K823005, K823008 & K823013 picked & inoculated into LB-medium
Incubated @ 37 °C, 250 rpm

Miniprep

Prep the Colonies of the ON

From now on:
K823005: BB04
K823008: BB05
K823013

15/06/17

PCR

	PCR9	PCR10
0,5ul template	BB K8230008	GFP4 (from PCR6)
1ul Primer	iILS8	iILS9
1ul Primer	iILS2	iILS5
10ul	buffer	buffer
4ul	dNTPs	dNTPS
2ul	Polymerase	Polymerase
31,5ul	H2O	H2O

Gel electrophoresis

Gel of Digest of the 06/16
Lane1: GFP04, Lane 2: BB08

Gel Extraction

Extraction of BB08 and GFP04

15/06/18

CPEC

CPEC2 (pILS5)

	CPEC2
2,5ul	BB08
4,29ul	GFP04
27,21	H2O
10ul	buffer
4ul	dNTPs
2ul	Polymerase

15/06/24

PCR

PCR for pILS4 and pILS6

PCR Mix	PCR7	PCR8	PCR11	PCR12
1ul	BB05	GFP	BB13	GFP
1ul	Primer iILS2	Primer iILS5	Primer iILS18	Primer iILS5
1ul	Primer iILS16	Primer iILS15	Primer iILS115	Primer iILS17
31ul	H2O	H2O	H2O	H2O
10ul	buffer	buffer	buffer	buffer
4ul	dNTPS	dNTPS	dNTPS	dNTPS
2ul	Polymerase	Polymerase	Polymerase	Polymerase

Gel electrophoresis

Load the PCR on a gel to cut out

Gel Extraction

Extract all PCRs

15/06/29

CPEC

pILS4		pILS6
BB04	0,2ul	BB06	0,8ul
GFP4	1,2ul	GFP6	0,9ul
H2O	32,62ul	H2O	32,35ul
buffer	10ul	buffer	10ul
dNTPs	4ul	dNTPs	4ul
Polymerase	4ul	Polymerase	4ul

Next day: Transformation - no colonies grew

15/07/01

PCR

PCR for pILS5

PCR Mix	PCR9	PCR10
0,5ul	BB08	GFP
1ul	iILS8	iILS9
1ul	iILS2	iILS5
31,5ul	H2O	H2O
10ul	buffer	buffer
4ul	dNTPs	dNTPs
2ul	Polymerase	Polymerase

PCR

PCR of GFP4, GFP6, BB4, BB6
Gel showes that BB6 was not succesful

Gel Extraction

extract BB4, GFP4, GFP6 from gel

PCR

Repeat PCR of BB6

15/07/02

CPEC

CPEC5		CPEC4
BB4	3,5ul	BB04	1,9ul
GFP5	5,1ul	GFP4	2,2ul
H2O	25,4ul	H2O	29,9ul
buffer	10ul	buffer	10ul
dNTPs	4ul	dNTPs	4ul
Polymerase	4ul	Polymerase	4ul

Transformation chemocompetent

Transform CPECS into NEB (chemically)
no colonies grew

15/07/06

CPEC

CPEC5		CPEC4
BB4	3,5ul	BB04	1,9ul
GFP5	5,1ul	GFP4	2,2ul
H2O	25,4ul	H2O	29,9ul
buffer	10ul	buffer	10ul
dNTPs	4ul	dNTPs	4ul
Polymerase	4ul	Polymerase	4ul

use different cycler protocol

15/07/07

Transformation chemocompetent

Transform CPEC4, 5 into NEB turbo (chemical transformation)

Glycerol stocks

Prepare glycerol stocks (siGEM08, -siGEM11) of pILS4,5,6 of overnight cultures

Miniprep

Miniprep of Overnight Cultures

PCR

Repeat PCR7-12

15/07/08

PCR

PCR of 7-12
PCR7,8,9,10,12 were successful
Repeat PCR11

Gel Extraction

Extract PCRs that worked

CPEC Standard

CPEC4		CPEC6
BB4	0,41ul	BB06	0,37ul
GFP4	4,23ul	GFP6	9,26ul
H2O	29,35ul	H2O	24,37ul
buffer	10ul	buffer	10ul
dNTPs	4ul	dNTPs	4ul
Polymerase	4ul	Polymerase	4ul

Transformation chemocompetent

Transform PEC5 and CPEC6 of the 6th into NEB chem comp. Use 5ul of DNA.
No colonies for CPEC4 the next day
Colonies for CPEC5.

Inoculation

Inoculate 8 colonies of CPEC5 in 5ml LB-CAM and incubate @37°C for 6h.

Miniprep

Prep the cultures.

Analytical digest

Digest the 8 plasmids with PvuII

15/07/10

Gel Extraction

Extract PCR BB6, BB4, GFP4 and GFP6 out of Gel

CPEC

CPEC4		CPEC6
BB4	1,25ul	BB06	8ul
GFP4	0,42ul	GFP6	0,42ul
H2O	32,33ul	H2O	31,58ul
buffer	10ul	buffer	10ul
dNTPs	4ul	dNTPs	4ul
Polymerase	4ul	Polymerase	4ul

PCR

New PCR of BB6

Gel Extraction

Extract BB6 BB6

CPEC

CPEC4		CPEC6
BB4	2,4ul	BB06	1,3ul
GFP4	0,7ul	GFP6	0,8ul
H2O	30,9ul	H2O	31,9ul
buffer	10ul	buffer	10ul
dNTPs	4ul	dNTPs	4ul
Polymerase	4ul	Polymerase	4ul

15/07/11

Transformation chemocompetent

Transform all previous CPECs into NEB turbo, chemical competent cells

15/07/12

Inoculation

Pick 8 colonies of each construct and grow them in LB CAM

15/07/13

Miniprep

Prep all the colonies from construct 4 and 6

Analytical digest

Digest with PvuII

ILS — **Figure 6:** Lower bands look ok, but somehow there are additional long bands. Chromosomal DNA? Repeat Miniprep

15/07/15

Miniprep

Repeat Miniprep of colony 1-8 of construct 4 and 6

Digest

Repeat digest with PvuII

Overnight Cultures

Start ONC of 6.1 and 6.6

15/07/16

Digest

New digest with new Minipreps with PvuII

ILS — **Figure 7:** Loaded also undigested plasmid; still weird long bands

ILS — **Figure 8:** Loaded also undigested plasmid; still weird long bands

ILS — **Figure 9:** Loaded also undigested plasmid; still weird long bands

Over night cultures

Start ONC of pILS5 for glycerol stock, 4.2, 4.4, 4.7, 6.1, 6.5

15/07/17

Glycerol stock

Prepare a glycerol stock of pILS5 (sIGEM20)and pILS4 (sIGEM21)

Transformation chemocompetent

Transform pILS4 into Dh5alpha

Miniprep

Prep 6.1 and 6.6

Digest

Analytical digest of 6.1 and 6.6 with PvuII

Sequencing

Send 6.1 and 6.6 for sequencing

15/07/20

Transformation chemocompetent

Transform pILS4,5,6 into wt3110 and MG1655

15/07/21

Over night culture

Pick colonies from pILS4,5,6 in wt3110 and MG1655 and start cultures

15/07/22

Glycerol stocks

WT3110: pILS4-6 - sIGEM 25-27
MG1655: pILS4-6 - sIGEM 28-31

15/07/27

Restreak all constructs

Restreak all strains on plates from glycerol stock

15/08/11

Transformation chemocompetent

Transformation of E1010 and J23100 into NEB turbo
J23100 wasn't succesful

15/08/12

FACS

Measurement of 3 biological replicats and 3 technical replicats

t=	dilution
0h	-
1h	-
2h	1:10
3h	1:100
4h	1:100
5h	1:100
6h	1:100
6h	1:100

15/08/13

Transformation chemocompetent

Repeat Transformation of J23100 in NEB turbo

15/08/17

Transformation chemocompetent

Repeat Transformation of J23100 in NEB turbo

PCR

PCR of BB7, BB8, BB9, BB10, T10, BB11, T11, BB12, T12

Over night culture

ONC of E1010, pILS4, pILS5, pILS5 in DH5alpha

15/08/18

Miniprep

Prep the cultures of E1010, pILS4, pILS5 and pILS6

PCR

PCR of RFP7, RFP8, RFP9, RFP10, RFP11, RFP12

Gel of PCRS

DpnI digest

Add 2 ul of DpnI to each PCR tube, incubate for 30min @37°C

PCR Purification

Purify BB7-9 and RFP7-9
Low in concentration afterwards

Gel Extraction

Extraction of BB10, RFP10, Term10, BB11, RFP11, Term11, BB12, RFP12, Term12
Low concentrations: repeat PCRs

PCR

Repeat PCRs of BB7-12, RFP7-12, T10-12

Analytical gel

Check BB7, RFP7, BB8, RFP8, BB9, RFP9 on gel

DpnI digest

Add 2 ul of DpnI to each PCR tube, incubate for 30min @37°C

Transformation electrocompetent

Transform J23100 into electrocompetent cells

15/08/19

PCR Purification

Purify BB7, RFP7, BB8, RFP8, BB9, RFP9

Gibson Assembly

15min @50°C

	BB	RFP
7	0,58ul	0,91ul
8	0,8ul	0,87ul
9	0,68ul	0,74

Transformation electrocompetent

Transform the Gibson Assemblies into NEB turbo

PCR

PCRs of BB10-12, RFP10-12 and Term 10-12

Gel Extraction

Extract BB10, RFP10, Term 10, BB11, RFP11, Term 11, BB12, RFP12, Term 12

Over night culture

Start cultures of pILS4-6 in DH5alpha and MG1655

15/08/20

Platereader experiment

Grow pILS4-6 in MG1655 and DH5alpha in M9 inplater reader
Starting OD 0,01 in 96well plate, measure OD and GFP every 15min

Over night cultures

Start cultures of pILS7.1-7.8, pILS7.1-8.8, pILS7.1-9.8 and J23100

Transformation electrocompetent

Transform pILS9 into NEB turbo

15/09/21

Miniprep

Prep the cultures of pILS7.1-7.8, pILS8.1-8.8 and pILS9.1

PCR Standard

Make the PCRS of Promoter 10-12

Gel

no bands visible at expected length

15/08/22

Digest

Digest pILS7.1-7.8, pILS8.1-8.8 and pILS9.1 with PvuII

**Figure 11:** pILS7.1, 8.3 and 9.1 look promising, send for sequencing

15/08/24

Over night cultures

Start cultures of pILS9.2, pILS9.3, pILS9.4 and pILS9.5

Transformation electrocompetent

Transform 0040, I20270 into DH5alpha and pILS9 (Gibson) in NEB turbo

15/08/25

Restreak from glycerol stock

Restreak 0040, I0270, pILS4, pILS5, pILS6 and DH5alpha on plates from glycerol stock - incubate for 16h

Digestion of Promoter/h2> Digest Promoter 10-12 with PvuII and EcoRI

Gel Extraction

extract the band

PCR

make a PCR on gel extracted digested promoter
- no bands visible

15/08/26

Over night cultures

Start cultures (8 biological replica) of 0040, I0270, pILS4, pILS5, pILS6 and DH5alpha

Sequencing

Sequencing of pILS7-9 showed point mutations

15/08/27

Measurement with platereader for ILS submission

Dilution of each culture to OD of 0.5 (platereader) and measurement of fluorescence intensities

15/08/28

Measurement with platereader for ILS submission

Dilution of each culture to OD of 0.5 (cuvette) and measurement of fluorescence intensities

15/08/29

PCR

Amplify parts for pILS7-9

PCR Purification

Purification of the PCRs

Gibson Assembly

Assembly of pILS7-9

Transformation electrocompetent

Transform Gibson Assembly into NEB Turbo

15/08/30

Over night cultures

Start cultures of Gibson Assemblies and J23100

15/08/31

Miniprep

Prep the cultures of pILS7-9, J23100

Sequencing

Send pILS7-9 for sequencing:
Constructs: 2.7.2, 2.8.2, 2.9.3, 3.7.1, 3.7.2, 3.8.1, 3.8.3, 3.9.2, 3.9.3

PCR

Fuse two primers to promoter by PCR

	Parts
5ul	iILS30
5ul	iILS31
2ul	Polymerase
4ul	dNTPs
10ul	buffer

PCR Purification

Purify the fragment of the Primer PCR

CPEC

	10.1	10.3	11.1	11.3	12.1	12.3
BB	0,7ul	0,7ul	0,65ul	0,65ul	1,14ul	1,14ul
RFP	0,32ul	0,32ul	0,208ul	0,208ul	0,26ul	0,26ul
Promoter	0,32ul	0,46ul	0,32ul	0,46ul	0,32ul	0,46ul
Terminator	3,81ul	3,81ul	3,81ul	3,81ul	3,81ul	3,81ul
H2O	28,85ul	28,71ul	28,94ul	28,81ul	28,48ul	28,34ul
dNTPS	4ul	4ul	4ul	4ul	4ul	4ul
Buffer	10ul	10ul	10ul	10ul	10ul	10ul
Polymerase	2ul	2ul	2ul	2ul	2ul	2ul

15/09/01

Sequencing

Sequencing of pILS2.8.2 and pILS3.7.1 were successful
For pILS9 still no positive clone - send more samples for sequencing

	10.4	11.4	12.4
BB	4,17ul	17,13ul	14,43ul
RFP	7,33ul	7,33ul	7,33ul
Promoter	0,59ul	0,59ul	0,59ul
Terminator	0,56ul	0,56ul	0,56ul
H2O	21,56ul	7,8ul	11,08ul
dNTPS	4ul	4ul	4ul
Buffer	10ul	10ul	10ul
Polymerase	2ul	2ul	2ul

Sequencing

Send pILS2.9.1 and 2.9.2 for sequencing

Over night cultures

Start cultures for 9, 10.1, 11.1, 12.1

15/09/03

Miniprep

Prep ONC 9+10+10

Analytical Digest

Digest with PvuII

Sequencing

Sequencing of 12.3, 11.3, 10.3, 9.3

15/09/07

Sequencing

10.3.1 was correct -> pILS10
11.3.1 was correct -> pILS11
Transformation of pILS10, pILS11 in DH5alpha cells

PCR

	PCR 9.1	PCR 9.2	PCR 12.1	PCR 12.2
0,5 ul Template	pILS11	pILS6	pILS6	pILS8
1ul	Primer ILS 17	Primer ILS 18	Primer ILS 21	Primer ILS 24
1ul	Primer ILS 20	Primer ILS 22	Primer ILS 22	Primer ILS 20
31,5 ul	H2O	H2O	H2O	H2O
dNTPS	4ul	4ul	4ul	4ul
Buffer	10ul	10ul	10ul	10ul
Polymerase	2ul	2ul	2ul	2ul

DpnI-Digest

1ul Enzyme, 30 min at 37°C

after the Thermo Fischer PCR-Clean up Kit Protokoll

Gibson Assembly

Assemble PCRs via Gibson

Transformation electrocompetent

Transform Gibson into NEB turbo

Over night culture

of pILS4,5,6 in DH5alpha for quantitiative proteomics

15/09/08

Over night culture

of pILS7,8 in DH5alpha

Over night culture

of pILS9,12 from Gibson Assembly

15/09/09

Glycerol stocks

pILS 7 in DH5alpha -> SiGEM 51
pILS 8 in DH5alpha -> SiGEM 52

Miniprep

of ONC 9,12 from Gibson Assembly

Analytical-Digest of 9,12

1ul PvuII, 30 min at 37°C

Sequencing

of pILS9,12 from Gibson Assembly

Over Night Cultures

of pILS4,5,6

15/09/10

Start of Evolution Study

ONC of pILS 4,5,6 in DH5alpha in 3 biologcal replica

Preperation of samples 4,5,6 for Proteomics

Sonication of ONC pILS4,5,6 for 15s, 15 Cycles
-> to Uwe Linne