Team:Marburg/Labbook/InterLab

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'
ILS
Figure 1: PCR1, 2, 3, 4


PCR

PCR of GFP (PCR2,4,6)

Gel electrophoresis

PCR2,4,6
ILS
Figure 2: PCR1, 2, 3


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

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

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


15/07/12

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



15/07/13

Transformation

Repeat Transformation of J23100 in NEB turbo


15/07/17

Transformation

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

ILS
Figure 10: Weird bands

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

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

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

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

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
ILS
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

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

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

Transformation

Transformation of CPEC10.1-10.4, 11.1-11.4 and 12.1-12.4 into NEB (with 1ul and 2ul)

PCR

Repeat PCR of primer bindin iILS30, iILS31

PCR Purification

Purify the Primer PCR

CPEC

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



15/09/02

Transformation

Trafo of constr. 10,4+11,4+12,4 in NEB Turbo

PCR

PCR of iILS30+iILS31

PCR Cleanup

PCR-Clean up of iILS30+iILS31

CPEC

CPEC of construct 5

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 of ONC 9+10+10

Analytical 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

New Method for 9/12

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



dNPI-Digest

1ul Enzyme, 30 min at 37°C

after the Thermo Fischer PCR-Clean up Kit Protokoll

Gibson Assembly

Transformation in NEB turbo cells



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

Preperation of Glycerolstock

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 Linner

Preperation of samples 4,5,6 for Microscopy

measured with Oliver and Anne


15/09/11

8am: FACS measurement

2ml Tethering Buffer +100 ul Sample
afterwards dilued to an OD of 0,05
Incubated at 30° for 12h

8pm: FACS measurement

2ml Tethering Buffer +100 ul Sample
afterwards dilued to an OD of 0,05
Incubated at 30° for 12h


15/09/12

8am: FACS measurement

2ml Tethering Buffer +100 ul Sample
afterwards dilued to an OD of 0,05
Incubated at 30° for 12h

8pm: FACS measurement

2ml Tethering Buffer +100 ul Sample
afterwards dilued to an OD of 0,05
Incubated at 30° for 12h

Transfomation

of piLS7,8,10,11, J0445, J20270 in DH5alpha and MG1655


15/09/13

8am: FACS measurement

2ml Tethering Buffer +100 ul Sample
afterwards dilued to an OD of 0,05
Incubated at 30° for 12h

8pm: FACS measurement

2ml Tethering Buffer +100 ul Sample
afterwards dilued to an OD of 0,05
Incubated at 30° for 12h


DATE

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