Team:Potsdam Bioware/Lab/Labjournal/October

Contents 1 AID 1.1 2012-10-10 1.2 2012-10-11 1.3 2012-10-26 2 Antibody 2.1 2012-10-12 2.2 2012-10-15 2.3 2012-10-17 2.4 2012-10-19 2.5 2012-10-20 2.6 2012-10-21 2.7 2012-10-22 2.8 2012-10-22 2.9 2012-10-23 2.10 2012-10-24 2.11 2012-10-25 3 Virus 3.1 2012-10-19 3.2 2012-10-20

AID

2012-10-10

Inoculation of plasmid samples of the 48h retransformation plates (after FACS)

Investigators: Tom

Time: 2012-10-10

Materials:

• LB medium
  

• Amp stock solution
  

• plate with cultures:
  

EGFR-C-AID without NES, with NLS+Kozak sequence+eGFP

(after FACS selection)

Method:

Inoculation of:

5 cultures per plate in 5 ml LB medium + 5µL Amp.

(--> 5 cultures)

Further tasks:

• Miniprep
  

2012-10-11

Purification of Retrafo plasmids

Investigators: Rico

Results: inoculation was not successful

2012-10-26

Purification of transfected wt AID, modified AID, TAL-AID plasmids cotransfected with small antibody construct and transformation

Investigators: Rico, Stefan, Tom

Method: transfected cells were lysed, plasmids purified and transformed into E. coli cells

Antibody

2012-10-12

cell culture

Investigator:Kerstin

Topic: cell-culture

• passaging of CHO w Zeocin
• passaging of HT1080
• passaging of HEK AAV 293
• passaging of HeLa

Topic: planning new primer for integration of new RAGE-transmembrane domain and RAGE-signalpeptide

Investigator: Sascha

Materials and Methods: Geneious

Topic: Primer design and ordering for integration of RAGE-transmembrane domain into scFVconstruct and nanobody-geneart construct

Investigator: Sascha

Materials and Methods: Geneious

Results:

• scFv:startprimer-fw
  • GCCTCTAGAGCTAGCATGGCAGC
• rvp:scfv+overhang to TEV/TMD
  • CGCCCAAGATTCCCAGGGCCAGGGCGAGGGTGCCGCTTCCGCCGCCACCGCTTCCCCCTTGAAAATATAAATTCTCTCCAGATCCCCGTTT

GATCTCCAGTTCTGTCC

• fwp:nterm. of yfp to TMD
  • CCCTGGGAATCTTGGGCGGCCTGGGGACCGCTGCCCTCTTGATCGGCGTGATCCTGTGGCAGAGAAGGTCTGGAGTGAGCAAGGGCGAG

GAGC

• scFv:endprimer-rv
  • GAGCTGCAGCGGCCG
• rage:startprimer-fw_nano/Fc
  • CTTGAATTCGCGGCCG
• rage-rv:Fc to rageTMD
  • CGCCCAAGATTCCCAGGGCCAGGGCGAGGGTGCCGCTTCCTAATAACTTCGTATAATGTATGCTATACGAAGTTATCCC
• rage-fwp:mcherry/rageTMD
  • CCCTGGGAATCTTGGGCGGCCTGGGGACCGCTGCCCTCTTGATCGGCGTGATCCTGTGGCAGAGAAGGGGCTCCATGGTGTCCAAGG
• rage:endprimer in mcherry/loxp
  • AAGTCACTGCAGCGGCC

2012-10-15

cell culture

Investigator:Kerstin

Topic: cell-culture

• passaging of CHO w Zeocin
• passaging of HT1080
• passaging of HEK AAV 293
• passaging of HeLa

2012-10-17

cell culture

Investigator:Kerstin

Topic: cell-culture

• passaging of CHO w Zeocin
• passaging of HT1080
• passaging of HEK AAV 293
• passaging of HeLa
• passaging of stably transfected Clones 4.1, 4.2, 4.3, 4,4, 29.1, 29.2, 29.3 with 550µg/ml Hygromycin

2012-10-19

cell culture

Investigator:Stefan/Kerstin

Topic: cell-culture

• passaging of CHO w Zeocin
• passaging of HT1080
• passaging of HEK AAV 293
• passaging of HeLa

PCR: amplification of EYFP and mcherry with overhang of new RAGE-TMD

Investigators: Sascha
Materials:

• Template: EYFP_Bba_E0030 and nano body-geneart-construct
• Phusion-polymerase
• 10x Phusion buffer HF
  
• dNTPs (10mM)
• eYFP: fwp:nterm. of yfp to TMD; primerVI: c-terminus of eyfp w
• mcherry: rage-fwp:mcherry/rageTMD; rage:endprimer in mcherry/loxp
• Thermocycler
  

Methods: 2x master mix of each: eYFP and mcherry

reagent	volume [µL]
10x Phusion HF buffer	20
dNTPs	2
fwp:nterm. of yfp to TMD; rage-fwp:mcherry/rageTMD	5
primerVI: c-terminus of eyfp w; rage:endprimer in mcherry/loxp	5
template EYFP (10ng/µl); template nanobody-geneart-construct (10ng/µl)	2
Phusion Polymerase	1,0
water	66

Program

step	Temperature [°C]	duration [s]	cycles
initial denaturation	98	30	1
denaturation	98	8	30
annealing	gradient: 64,7°C/71°C for eYFP; 62,0°C/66,1°C for nanobody-geneart-construct	10	30
elongation	72	15	30
final elongation	72	600	1
cooling	8	∞	1

Topic: preparative gelelectrophoresis of amplified EYFP and mcherry with RAGE-TMD and RAGE-signalpeptide overhangs

Investigator: Sascha
Materials:

• agarose
• 1xTAE-buffer
• 10xFD Green Buffer
  

Method:

• 1% agarosegel, 55ml
• 120V

Results:

• amplified eYFP and mcherry showed sufficient bp-size after PCR
  

Further Tasks:

• gel extraction and concentration measurement
• amplification of scFv-fragment and nanobody/Fc

gel extraction of amplified eYFP and mcherry out of 1% agarosegel

Investigators:Sascha
Results:
[eYFP] =
[mcherry] =
Further Taks:

• extension PCR of scFv and nanobody

2012-10-20

PCR: amplification of nanobody/Fc with overhang of new RAGE-TMD

Investigators: Sascha
Materials:

• Template: nanobody-geneart-construct
• Phusion-polymerase
• 10x Phusion buffer GC
  
• dNTPs (10mM)
• nanobody-geneart-construct: rage:startprimer-fw_nano/Fc; rage-rv:Fc to rageTMD
• Thermocycler
  

Methods: 2x master mix of each: eYFP and mcherry

reagent	volume [µL]
10x Phusion BC buffer	20
dNTPs	2
rage:startprimer-fw_nano/Fc	5
rage-rv:Fc to rageTMD	5
template nanobody-geneart-construct (10ng/µl)	6
Phusion Polymerase	1,0
water	92

Program

step	Temperature [°C]	duration [s]	cycles
initial denaturation	98	30	1
denaturation	98	8	30
annealing	520°C/62°C/65,5°C	10	30
elongation	72	15	30
final elongation	72	600	1
cooling	8	∞	1

Topic: preparative gelelectrophoresis of amplified nanobody/Fc with RAGE-TMD overhangs

Investigator: Sascha
Materials:

• agarose
• 1xTAE-buffer
• 10xFD Green Buffer
  

Method:

• 1% agarosegel, 55ml
• 120V

Results:

• amplified nanobody/Fc showed sufficient bp-size @ 1252bp
  

Further Tasks:

• gel extraction and concentrations measurement

gel extraction of amplified eYFP and mcherry out of 1% agarosegel

Investigators:Sascha
Results: [nanobody/FC] = 2,5ng/µl</b> Further Taks:

• extension PCR of scFv and nanobody
• assembly-PCR of nanobody/Fc with mherry via RAGE-transmembrane domain

PCR: assembly-PCR of nanobody/Fc with mcherry over RAGE-TMD

Investigators: Sascha
Materials:

• Template: nanobody/FC and mcherry with TMD-overhangs
• Phusion-polymerase
• 10x Phusion buffer GC
  
• dNTPs (10mM)
• Thermocycler
  

Methods: 2x master mix of each: eYFP and mcherry

reagent	volume [µL]
10x Phusion BC buffer	20
dNTPs	2
rage:startprimer-fw_nano/Fc was added after 15 assembling cycles	5
rage:endprimer in mcherry/loxp was added after 15 assembling cycles	5
templates: nanobody/FC with TMD-overhangs ; mcherry with TMD-overhangs (10ng/µl)	2µl of mcherry and 8µl of nanobody/Fc
Phusion Polymerase	1,0
water	57

Program

step	Temperature [°C]	duration [s]	cycles
initial denaturation	98	30	15
denaturation	98	10	15
annealing	60°C/70°C	12	15
elongation	72	38	15
initial denaturation	98	30	15
denaturation	98	10	15
annealing	60°C/70°C	12	15
elongation	72	38	15
final elongation	72	600	1
cooling	8	∞	1

Topic: analytical gelelectrophoresis of amplified assembled nanobody/Fc with RAGE-TMD overhangs

Investigator: Sascha
Materials:

• agarose
• 1xTAE-buffer
• 10xFD Green Buffer
  

Method:

• 1% agarosegel, 55ml
• 120V

Results:

• assembled nanobody/Fc-RAGE-TMD-mcherry showed sufficient bp-size: 2,1kb
  

Further Tasks:

• PCR-Clean up of 60°C-assembling PCR-mix
• amplification of assembeled nanobody/Fc-RAGE-TMD-mcherry

PCR-Clean up of preparative gel electrophoresis of assembled nanobody/Fc-RAGE-TMD-mcherry and concentration measurement

Investigator: Sascha
Materials:

• NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)
  

Methods:

• according to CleanUp-protocol

Results: [assembled nanobody/Fc-RAGE-TMD-mcherry ] =
Further Taks:

• preparative gel electrophoresis of assembled nanobody/Fc-RAGE-TMD-mcherry

2012-10-21

Topic: preaparative gel electrophoresis of assembled nanobody/Fc-RAGE-TMD-mcherry

Investigator: Stefan
Materials:

• agarose
• 1xTAE-buffer
• 10xFD Green Buffer
  

Method:

• 1% agarosegel, 55ml
• 120V

Results:

• assembled nanobody/Fc-RAGE-TMD-mcherry showed sufficient bp-size: 2,1kb
  

Further Tasks:

• gel extraction
• cloning into pcDNA5FRT

2012-10-22

cell culture

Investigator:Kerstin

Topic: cell-culture

• passaging of CHO w Zeocin
• passaging of HT1080
• passaging of HEK AAV 293
• passaging of HeLa
• transfection of stably transfected clone 29.1 with Cre Recombinase
• seeding of HT1080 in Ibidi Dish for infection with virus

2012-10-22

gel extraction of assembled nanobody/Fc-RAGE-TMD-mcherry out of 1% agarosegel

Investigators:Sascha
Results: [assembled/amplified nanobody/Fc-RAGE-TMD-mcherry] = 60,4ng/µl
Further Taks:

• cloning into pcDNA5FRT

preparative digestion of assembled/amplified nanobody/Fc-RAGE-TMD-mcherry with NheI and ApaI

Investigator: Sascha
Materials:

• Fast Digest NheI
• Fast Digest ApaI
• 10x FD Green Buffer
• assembled/amplified nanobody/Fc-RAGE-TMD-mcherry
• sterile water

Method:
2x

• 20µl pcdna5frt (60,4ng/µl)
• 2µl NheI
• 2µl ApaI
• 3µl 10x FD Green Buffer
• 4µl sterile water
• digestion for 1h at 37°C
  

>Results:
Further Tasks:

• PCR-Clean Up

PCR-Clean up of digested assembled/amplified nanobody/Fc-RAGE-TMD-mcherry and concentration measurement

Investigator: Sascha
Materials:

• NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)
  

Methods:

• according to CleanUp-protocol

Results: [assembled/amplified nanobody/Fc-RAGE-TMD-mcherry] = 6ng/µl
Further Taks:

• ligation with pcDNA5FRT (dephos +dig)

ligation of digested dephosporylated pcDNA5FRT (NheI+ApaI) and digested assembled/amplified nanobody/Fc-RAGE-TMD-mcherry (NheI+ApaI)

Investigator: Sascha
Materials:

• ligation calculator: http://www.insilico.uni-duesseldorf.de/Lig_Input.html
• T4 DNA ligase
• ligase buffer
• digested geneart-nanobody-construct
• digested and dephosporylated pcDNA5FRT

Method: ligation-ratio--> 1:3
:

• 1µl T4 DNA ligase
• 2µl T4 DNA-ligase buffer
• 15 ng dig. pcDNA5FRT
• 15 ng insert (assembled/amplified nanobody/Fc-RAGE-TMD-mcherry)
• sterile water ad 20µl
  

• 1:0 religation; same mix without insert

Further Tasks:

• transformation of ligation mix into XL1-blue competent E. coli cells

• incubation for 60min at RT

Further Tasks:

• transformation of ligation mix into XL1-blue competent E. coli cells

Transformation of ligated pcDNA5FRT- assembled/amplified nanobody/Fc-RAGE-TMD-mcherry into new XL1-blue competent E. coli cells

Investigator: Sascha
Materials:

• Bunsen Burner, Agar Plate with Ampicillin, 37 °C thermo mixer, centrifuge,
  
• 12 µl of each ligation mix
• icebox
• new competent E. coli cells (XL 1)

Method:

• according to manual
• 20µl of resuspended cell-suspension were plated on a LB-Amp-plate
• incubation o.n. at 37°C

Further Tasks:

• picking clones

PCR: amplification of scFv with overhang of new RAGE-TMD

Investigators: Sascha
Materials:

• Template: scFv_Bba pks…bla
• Phusion-polymerase
• 10x Phusion buffer HF
  
• dNTPs (10mM)
• rvp:scfv+overhang to TEV/TMD; primerI-fw:rage-signal, xbaI,
• Thermocycler
  

Methods: 3x master mix of each: scFv

reagent	volume [µL]
10x Phusion HF buffer	30
dNTPs	3
scfv+overhang to TEV/TMD	5
primerI-fw:rage-signal, xbaI	5
template scFv_Bba pks…bla (10ng/µl)	3
Phusion Polymerase	1,5
water	195

Program

step	Temperature [°C]	duration [s]	cycles
initial denaturation	98	30	1
denaturation	98	8	30
annealing	53°C/60°C/71°C	10	30
elongation	72	15	30
final elongation	72	600	1
cooling	8	∞	1

Topic: preparative gel electrophoresis of amplified scFv with RAGE-TMD overhangs

Investigator: Sascha
Materials:

• agarose
• 1xTAE-buffer
• 10xFD Green Buffer
  

Method:

• 1% agarosegel, 55ml
• 120V

Results:

• amplified scFv showed sufficient bp-size @ 9102bp
  

Further Tasks:

• PCR-Clean Up of amplified scFv

gel extraction of amplified scFv-RAGE-TMD out of 1% agarosegel

Investigators:Sascha
Results: [amplified scFv-RAGE-TMD] = 60,4ng/µl
Further Taks:

• assembling with eYFP-RAGE overhang

PCR: assembly-PCR of scFv with eYFP over RAGE-TMD

Investigators: Sascha
Materials:

• Template: scFv with eYFP with TMD-overhangs
• Phusion-polymerase
• 10x Phusion buffer GC
  
• dNTPs (10mM)
• Thermocycler
  

Methods: 3x master mix of each: eYFP

reagent	volume [µL]
10x Phusion GC buffer	30
dNTPs	3
scFv:startprimer-fw, was added after 15 assembling cycles	7,5
scFv:endprimer-rv was added after 15 assembling cycles	7,5
templates: scFv with TMD-overhangs and RAGTE-signalpeptide (10ng/µl); eYFP with TMD-overhangs (10ng/µl)	1µl of eYFP and 1µl of scFv
Phusion Polymerase	1,5
water	96,5

Program

step	Temperature [°C]	duration [s]	cycles
initial denaturation	98	30	15
denaturation	98	10	15
annealing	60°C/70°C	12	15
elongation	72	38	15
initial denaturation	98	30	15
denaturation	98	10	15
annealing	60°C/70°C	12	15
elongation	72	38	15
final elongation	72	600	1
cooling	8	∞	1

Topic: analytical gelelectrophoresis of assembled scFv-RAGE-eYFP construct

Investigator: Sascha
Materials:

• agarose
• 1xTAE-buffer
• 10xFD Green Buffer
  

Method:

• 1% agarosegel, 55ml
• 120V

Results:

• assembled scFv-RAGE-eYFP construct showed sufficient bp-size: 1,7kb
  

Further Tasks:

• PCR-Clean up of 60°C-assembling PCR-mix
• amplification of assembeled scFv-RAGE-eYFP construct

2012-10-23

cell culture

Investigator:Stefan

Topic: cell-culture

• seeding of CHO and HeLa in Ibidi Dishes for transfection with new Nanobody RAGE construct
• infection with Virus (CFP on surface and YFP as GOI, AAV with Sortase motif)

gelextracton of scFv-RAGE-TMD-EYFP in Flp-In vector construct out of 1% agarosegel

Investigators:Maria
Aim: gelextraction and preparation of cleaned scFv-RAGE-TMD construct
Materials:

• Gel-Clean-Up Kit

Method:

• according to manual

Results:

• 32,6 ng/µl

Further tasks:

• digestion with NheI and ApaI

preparative digestion of scFv-RAGE-TMD construct with NheI and ApaI

Investigator: Maria
Aim: digestion of scFv-RAGE-TMD construct with NheI and ApaI for ligation into Flp-in vector
Materials:

• Fast Digest NheI
• Fast Digest ApaI
• 10x FD Green Buffer
• scFv construct
• sterile water

Method:

• 18,4µl scFv construct
• 2µl NheI
• 2µl ApaI
• 3µl 10x FD Green Buffer
• 4,6µl sterile water
• digestion for 2,5h at 37°C

Further Tasks:

• PCR clean-up
• ligation into

PCR clean-up of scFv-RAGE-TMD construct

Investigator:Maria
Aim: cleaning of scFv-RAGE-TMD
Materials:

• PCR-Clean-Up Kit

Method:

• according to manual

Results:

• concentration of cleaned scFv-RAGE-TMD = 19,7 ng/µl

ligation of digested scFv-RAGE-TMD construct and dephosporylated Flp-In vector

Investigator: Maria
Aim: ligation of digested scFv construct with Flp-In vector

Materials:

• ligation calculator: http://www.insilico.uni-duesseldorf.de/Lig_Input.html
• T4 DNA ligae
• ligase buffer
• digested scFv construct
• digested and dephosporylated Flp-In vector

Method: ligation-ratio--> 1:3

• 1µl T4 DNA ligase
• 2,5µl T4 DNA-ligase buffer
• 3,3µl digested scFv (19,7ng/µl)
• 1µl digested Flp-In vector (60,4ng/µl)
• 12,2µl sterile water

• incubation for 1h at RT

Further Tasks:

• transformation of ligation mix into XL1-blue competent E. coli cells

Transformation of ligated scFv-Flp-In vector into new XL1-blue competent E. coli cells

Investigator: Sascha
Materials:

• Bunsen Burner, Agar Plate with Ampicillin, 37 °C thermo mixer, centrifuge,
  
• 10 µl of cFv-Flp-In vector
• icebox
• competent E. coli cells (XL 1)

Method:

• according to manual
• 20µl of resuspended cell-suspension were plated on a LB-Cm-plate
• incubation o.n. at 37°C

Further Tasks:

• picking clones

2012-10-24

cell culture

Investigator:Stefan/Kerstin

Topic: cell-culture

• Transfection of new Nanobody RAGE construct in CHO and HeLa
• seeding of CHO and HeLa in Ibidi Dishes for transfection with new scFv RAGE construct
• passaging of CHO w Zeocin
• passaging of HT1080
• passaging of HEK AAV 293
• passaging of HeLa

Endotoxin free preparation of nanobody/Fc-RAGE-TMD-mcherry

Investigator:Maria

Materials: endotoxin free Mediprep kit, overnight culture

Methods: according to manual

Results: <b/> clone 2: 880,3 ng/µl

Further tasks: transient and stable transfection of CHO cells

2012-10-25

cell culture

Investigator:Stefan/Kerstin

Topic: cell-culture

• transfection of new scFv RAGE construct
• transfection of TAL-AID from cooperation with Freiburg iGEM Team in CHO cells (co-transfection with clone 4)
• purification of Nanobody from supernatant of Cre recombinase transfected cells with magnetic beads
• Western Blot of purified Nanobody

Mini Prep of scFv-Flp-In vector clones

Investigator:Maria

Materials: overnight cultures scFv-Flp-In clones, Mini prep kit

Methods: according to manual

Results: Clon I: 514,8 ng/µl
Clon II: 673 ng/µl
Clon III: 639,7 ng/µl
Clon VI: 499 ng/µl

Further tasks: analytical digestion, analytical gelelectrophoresis

analytical digestion of ligated scFv-Flp-In construct

Investigators:Maria

Materials:

• all 4 prepared scFv-Flp-In clones
• FastDigest StuI, SpeI and PstI
• 10x FD Green Buffer
• sterile water

Method: I:

• 10µl mix: 1µl StuI, 1µl 10x FD Green Buffer, 1µl of each clone respectively (approximately 250 ng DNA), sterile water add to 10µl

II:

• 10µl mix: 1µl StuI, 1µl 10x FD Green Buffer, 1µl of each clone respectively (approximately 250 ng DNA), sterile water add to 10µl
• incubation at 37°C for 30 min

further tasks:

• analytical gelelectrophoresis

Gelelectrophoresis of analytical digested scFv-Flp-In construct

Investigators:Maria

Aim: checking plasmid-size after ligation of Flp-In vector with scFv-RAGE-TMD in 1% agarosegel
Materials:

• agarose
• 1xTAE-buffer
• 10xFD Green Buffer

Method:

• 1% agarosegel, 100ml
• 120 V

Results:

• ligation successful
  

Further Tasks:

• endotoxin free of clone

Endotoxin free preparation of scFv-RAGE-TMD

Investigator:Maria

Materials: endotoxin free Mediprep kit, overnight culture

Methods: according to manual

Results: <b/> clone 1: 2349,7 ng/µl

Further tasks: transient and stable transfection of CHO cells

Virus

2012-10-19

EGFRd3 purification

Investigator: Tobias/Xenia

Aim: Periplasma-Extract

Materials:

• E. coli with expressed EGFR
• Extraction buffer (50 mM Tris, 150 mM NaCl, 500 mM sucrose)
• loading buffer (50 mM Tris, 150 mM NaCl, 30 mM imidazol)

Method:

• resuspend E. coli with extraction buffer
• incubate 2 hours at 4°C
• centrifuge and take supernatant
• dialyze against loading buffer over night

Further tasks:

• purification with Ni-NTA

2012-10-20

EGFRd3 purification

Investigator: Tobias

Aim: Purification with Ni-NTA

Materials:

• protein extract in loading buffer (50 mM Tris, 150 mM NaCl, 30 mM imidazol)
• wash buffer (50 mM Tris, 150 mM NaCl, 30 mM imidazol)
• elution buffer (50 mM Tris, 150 mM NaCl, 250 mM imidazol)
• Ni-NTA column (1 mL volume)

Method:

• loading Ni-NTA column with protein extract
• wash column with wash buffer (10fold column volume)
• elution with elution buffer and seperate ca. every 1 ml
• control purity with SDS-PAGE

Results:

Further tasks:

• concentrate purified EGFR and ligate with sortase