Team:LMU-TUM Munich/Labjournal

Monday, May 16th

Streptavidin plasmids_control

Investigator: JB, LK, JH

Aim of the experiment: Verification of cloning

Procedure: * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen) (4 clones each of pSA1, pSAm1 in pASK75)

analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF), 1 µl SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O
5 µl on 1% agarose gel electrophoresis of digestion

Result: successful cloning verified, stored at -20 °C* 1. Lane: 5 µl Thermo Fisher, 1kb Ladder

2. to 9. Lane: 5 µl digestions of P6 to P13, band of SA (mut1) at about 300 bp, band of digested plasmid at about 3.000 bp

[[Image:|top]]

Streptavidin expression_trafo BL21

Investigator: JB, JH

Aim of the experiment: expression of pSA1 and pSAm1 in E. Coli BL21

Procedure: transformation according to protocol of P6 and P10 in competent E. Coli BL21

Result: plates (LB Amp) in incubator for further processing (37 °C)

Tuesday, May 17th

SDS Gel Analysis

Investigator: CG

Aim of the experiment: SDS gel analysis of collagen 1/2, eGFP, fraction 30 of egg-precipitation

Procedure: mixing of 80 µl samples with 20 µl SDS buffer and heating at 95°C for 10 min. 1 d staining, 1 d unstaining

Result:* 1. Lane: 8 µl Marker (Thermo Fisher #26610)

2. Lane: fraction 30 (IEC), 3 µl, band at 35 kDa, Avidin expected at 16 kDa
3. Lane: eGFP, 12 µl, band at 27 kDa eGFP expected at 27 kDa, many impurities
4. Lane: Collagen 1, 12 µl, no sharp band
5. Lane: Collagen 1, 12 µl, no sharp band

[[Image:|top]]

Minipreps pSb1C3-AviTag, -A3C5, pASK75-(SA1), -(SAm1)

Investigator: CR, CG

Aim of the experiment: Verification of cloning

Procedure: * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

analytic digestion with: 0,25 µl XbaI, 0,25 µl HindIII (HF) for pASK plasmids and 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 plasmids, 1 µl SmartCut Buffer, 5 µl Plasmid-DNA, 3,5 µl H2O

- 5 µl on 1% agarose gel electrophoresis of digestion

Result: successful cloning verified for pASK plasmids, repetition of pSb1C3 plasmids, stored at -20 °C* 1. Lane: 5 µl Thermo Fisher, 1 kb Ladder

2. Lane: 5 µl digestion of pSb1C3-AviTag
3. Lane: 5 µl digestion of pSb1C3-A3C5
4. Lane: 5 µl digestion of pASK75(SA1), EB elution
5. Lane: 5 µl digestion of pASK75(SA1), H2O elution
6. Lane: 5 µl digestion of pASK75(SAmut1), EB elution
7. Lane: 5 µl digestion of pASK75(SAmut1), H2O elution

[[Image:|top]]

Inoculation of pre-culture with BL21 (pASK75 (SA1)) in LB-medium

Investigator: CR

Aim of the experiment: Preculture for streptavidin expression in TB-medium

Procedure: * Add 50 µL ampicillin in 50 mL LB-medium

Picking colonies from BL21 (pASK75 (SA1))
Inoculate LB-medium
Incubate at 30°C over night

Wednesday, May 18th

Repetition of analytical gel of pSb1C3-AviTag, -A3C5

Investigator: CG, CR

Aim of the experiment: Verification of cloning

Procedure: * analytic digestion with: 0,25 µl NgomIV, 0,25 µl AgeI (HF) for pSb1C3 plasmids, 1 µl SmartCut Buffer, 8,5 µl Plasmid-DNA

10 µl on 2% agarose gel electrophoresis of digestion

Result:

[[Image:|top]]

Nächstes Mal 1kb ladder!!!!

Inoculation of BL21 (pASK75 (SA1)) culture in 2 L TB-Medium and induction of streptavidin production by addition of tetracycline

Investigator: CR

Aim of the experiment: Production of streptavidin

Procedure: * Ampicillin (2 mL) was added to the Medium (1:1000)

The pre-culture (50 mL) was poured into the Medium
Culture was incubated at 37°C and 140 rpm until OD₅₅₀ reached 0.5
To induce streptavidin expression anhydro-tetrazycline (200 µL) was added to the culture (1:10000)
The culture was incubated at 37°C and 140 rpm for 4 hours

Result:* Streptavidin expression by BL21

Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium

Investigator: CR, JB, JH

Aim of the experiment: Recombinant expression and purification of Streptavidin

Procedure: * After expression, cultures were transferred into centrifuge tubes and spun down in the centrifuge (4°C, 5000 rpm, 20 mins, F 4X1L rotor)

The supernatant was cast away and the pellet was transferred into a beaker of sufficient size and resuspended in fridge-cooled Tris Buffer B (50 mM Tris/HCl (pH = 8.0), 1 mM EDTA)
The solution was homogenized in the PANDA (ask supervisor)
The resulting lysate was transferred into centrifuge tubes and spun down (4°C, 18,000 rpm, 10 mins, XX34-rotor). The supernatant was cast away and the pellet was resuspended in 6M Gua-HCl (pH = 1.5) at 4°C overnight.

Dialysis of eGFP

Investigator: NA, JH, CR

Aim of the experiment: Purification of eGFP

Procedure: * eGFP was thawed on ice

eGFP was then poured in a dialysis hose (cut-off 14 kDa)
The hose was then placed in ice cold Tris/HCl 20 mM pH 8.0
The dialysis took place at 4°C over night

MiniPrep of quickchanged pNGAL146-A2

Investigator: NA

Aim of the experiment: Extraction of pNGAL146-A2 plasmid from XL1 blue

Procedure: * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

Sequencing of P14, P15 & P19

Investigator: CR, NA

Aim of the experiment: Sequencing of P14, P15 & P19

Procedure:

Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)

The different plasmids we prepared received the following barcodes:* P14 : FR11326653

P15 : FR11326655
P19 (K4): FR11326654
P16 (K1): FR11326652
P17 (K2): FR11326651
P18 (K3): FR11326650

Digestion of P16, P17, P18 & P19 with AgeI & HindIII + analytical gel

Investigator: NA, JH, CR

Aim of the experiment: Verification of success of quickchange

Procedure: * analytic digestion with: 0,25 µl HindIII (HF), 0,25 µl AgeI (HF), 1 µl SmartCut Buffer, 500 ng plasmid-DNA, fill up with ddH₂O (V_total= 10µL)

10 µl on 2% agarose gel for electrophoresis

Result:

[[Image:|top]]

No signal at 600 bp --> quickchange seems to be successful (waiting for sequencing)

Thursday, May 19th

Re-Sequencing of P19

Investigator: CR

Aim of the experiment: Re-Sequencing of P19

Procedure:

Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)

The different plasmids we prepared received the following barcodes:* P19 (K4): FR11326649

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: CG

Aim of the experiment: Re-Trafo of pSB1C3 RFP for later on: digestion, dephosphorylation and cloning

Procedure: transformation according to protocol of P4 E. Coli XL1

Result: plates (LB Cam) in incubator for further processing (37 °C)

Streptavidin refolding

Investigator: JB

Aim of the experiment: Refolding of denaturated Streptavidin

Procedure: After the pellet had almost completely dissolved in 6M GdmCl, the solution was spun down (4°C, 20 mins, 18,000 rpm). The supernatant was transferred carefully into a falcon tube and the pellet was cast away. Via a hydraulic pump (flow rate: 2x10 ml/min) the lysate was transferred Into 5L PBS 1x. Afterwards the pump was cleaned with technical isopropanol and ELGA water. The solution was stirred overnight at 4°C for refolding.

Biotinylation of BSA

Investigator: JB

Aim of the experiment: Biotinylation of BSA

Procedure: A 100 µM (=6.8 mg/ml) solution of BSA (Albumin fraction V, pH=7, in the fridge in the central lab) was created (V=10 ml). 220 µl of a 100 mM Biotin stock were added. The mixture was stored overnight Iin the fridge (4°C).

Result: Hopefully biotinylated BSA mixture in the fridge (4°C).

Friday, May 20^th

Qualitative analysis of streptavidin expression

Investigator: CG

Aim of the experiment: SDS gel analysis of recombinant strepatividin expression

Procedure: mixing of 80 µl sample with 20 µl SDS buffer and heating at 95°C for 10 min. 1 h staining, 1 night unstaining

Result: * 1. Lane: 8 µl Marker (Thermo Fisher #26610)

2. Lane: 12 µl culture aliquot, before induction
3. Lane: 12 µl culture aliquot, after induction
4. Lane: 3 µl culture aliquot of the lysed pelet, Strepatvidin expected at about 16 kDa
5. Lane: 3 µl culture aliquot of the supernatend after lysis, no Strepatvidin expected at about 16 kDa
6. Lane: 1.5 µl culture aliquot of the lysed pelet, Strepatvidin expected at about 16 kDa
7. Lane: 1.5 µl culture aliquot of the supernatend after lysis, no Streptavidin expected at about 16 kDa

[[Image:|top]]

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: LK

Aim of the experiment: Inoculation of pre-culture with E. coli XL1 (pSb1C3 -RFP) in LB-Chloramphenicol-medium

Procedure: * Picking of colonies for E. coli XL1 (pSb1C3 -RFP)

Inoculate in 5 ml LB-Chloramphenicol-medium
Incubate at 37°C over night at 200 rpm

Transformation of Biobricks in XL1-blue

Investigator: NA, JH

Aim of the experiment: Transformation

Procedure:

-10 µl dd H₂O in well of interest (standard distribution kit)

-1 µl Plasmid (out of well) to cells

-Transformation according to the SOP

Used bricks:

K577893, B0015, R0040, B0032, I14033, K747096

Ammonium sulfate precipitation of streptavidin

Investigator: JB

Aim of the experiment: Reduction of the protein solution volume and precipitation of streptavidin

Procedure: The 5 L protein solution was spun down (20 mins, 5,000 rpm) and the supernatant transferred into a beaker. In order to lower the volume of the solution for ammonium sulfate precipitation, the solution was first filtered via a membrane crossflow pump (membrane: Sartocon 0.45 µm, thick membrane).

Dialysis of biotinylated BSA

Investigator: NA

Aim of the experiment: purification of biotinylated BSA

Procedure: dialysis against Tris/HCl 20 mM, pH 8, 10 mM NaCl over night;

cut off : 14 kDa

Monday, May 23^rd

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: CR

Aim of the experiment: Cloning of A3C5 and Avi-Tag into pSB1C3

Procedure: * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

pSB1C3 was digested with AgeI and NgOMIV (10 µL plasmid + 1.5 µL AgeI + 1.5 µL NgoMIV + 3 µL CutSmart + 14 µL ddH₂O; incubation for 1h, 37°C)
FastAP was added and the reaction was incubated for another 2h at 37°C
The digestion was purified by gelelctrophoresis (1%, 75V, 1h)
Plasmid backbone was cut from the gel

Results:

[[Image:|top]]

Signal 1: linearized pSB1C3 (successfull digestion)

Signal 2: supercoiled pSB1C3 (digestion not successfull)

Signal 3: RFP-generator

--> next time gel should run longer (just if you need the back bone)

Filtration of Streptavidin and precipitation with Ammonium-sulfate

Investigator: MP, CR

Aim of the experiment: Concentration of Streptavidin

Procedure: * The solution was filtered via a membrane crossflow pump (membrane: Sartocon 0.45 µm, thick membrane).

The final volum was 0.5L
The solution was precipitated by addition of Ammonium-sulfate (2 steps: 1. 40% Ammonium-sulfate; 2. 70% Ammonium-sulfate)
After the first addition of ammonium-sulfate the solution was stirred (1h, 4°C)
Afterwards the solution was centrifuged (10.000 rpm; 30 min) and the supernatant was used for the second precipitation step
The solution was stirred again (over night, 4°C)

Inoculation of pre-culture with BioBricks in pSB1C3 in LB-Cam

Investigator: JH, JL

Aim of the experiment:

Procedure: * Add 50 µL chloramphenicol in 50 mL LB-medium

Picking colonies from K577893; B0015; B0032 (in pSB1C3) *
Inoculate 4 mL medium (CAM)
Incubate at 37°C over night

*Following cultures didn´t grow and were therefore not inoculated:

R0040; K747096; I14033

Retransformation of Biobricks in XL1-blue

Investigator: JH, JL, EF

Aim of the experiment: Transformation

Procedure: * 10 µl dd H₂O in well of interest (standard distribution kit)

1 µl Plasmid (out of well) to cells
Transformation according to the SOP
Used bricks:
K577893, B0015, R0040, B0032, I14033, K747096

Tuesday, May 24^th

Inoculation of pre-culture with BioBricks in pSB1C3 in LB-Cam

Investigator: CR

Aim of the experiment:

Procedure: * Add 50 µL chloramphenicol in 50 mL LB-medium

Picking colonies from K577893; B0015; B0032 (in pSB1C3) *
Inoculate 4 mL medium (CAM)
Incubate at 37°C over night

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: CR

Aim of the experiment: Gelextraction of Signal 1 from pSB1C3-digestion

Procedure: * Gelextraction was performed after manufacturer's protocol (Gelextractionkit, Qiagen)

Ammonium sulfate precipitation

Investigator: JB

Aim of the experiment: Precipitation the 70% saturated ammonium sulfate solution

Procedure: The ammonium sulfate solution, which actually was a suspension, was transferred into centrifuge tubes and spun down (10,000 rpm, 45 mins). As it turns out, unfortunately too much ammonium sulfate was used for the precipitation (wrong table). The precipitate was brought back into solution (20 mM Tris/HCl, pH 8.0) and loaded onto an SDS gel for analysis, along with other samples (flowthrough of the filtration from the day before).

MiniPrep of BioBricks in pSB1C3 (inoculated on May 23^th)

Investigator: JH

Aim of the experiment: Extraction of BioBricks B0032, B0015 and K577893 in pSB1C3 from XL1 blue

Procedure: * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

concentrations measured (in ng/µL):

(B0032) 360.2; (B0015) 254.9; (K577893) 99.2

Sequencing of P23, P24 & P24

Investigator: JH

Aim of the experiment: Sequencing of P23, P24 & P25 (x2)

Procedure:

Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (50-100 µM) and 2 µL sequencing primer)

The different plasmids we prepared received the following barcodes:* P23 (VF2) : FR11326647

P24 (VF2) : FR11326646
P25 (VF2) : FR11326645
P25 (VR) : FR11326644

Wednesday, May 25^th

Inoculation of pre-culture with tet-repressed GPF (P25) in LB-Cam

Investigator: NA

Procedure: * Add 50 µL chloramphenicol to 50 mL LB-medium

Picking colonies from plate (BL21)
Incubate over night at 37°C

=> dismissed, because plasmid sequence was inaccurate

MiniPrep of K747096, I14033, R0040, B0015, K577893 and B0032

Investigator: CR

Aim of the experiment: MiniPrep of BioBricks

Procedure: * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

Concentrations were measured (in ng/µL):

K747096: 286,2

I14033: 246,1

R0040: 243,5

B0015: 244,1

K577893: 440,9

B0032: 259,7

PCR of P19 with O3 and O4

Investigator: CR

Aim of the experiment: Amplification of quickchanged EspP

Procedure: *

reaction was performed with the Q5 High Fidelity DNA Polymerase (M0491, NEB) and after manufacturer's protocol (PCR Using Q5® High-Fidelity DNA Polymerase, NEB)
274,4 pg Plasmid were used for amplification (1 µL)
PCR program (standard):

Step	Temperature [°C]	Time [s]
initial Denaturation	98	30
35 cycles	98	10
72	30
72	60
final Extension	72	5
Hold	4	ꝏ

PCR product was purified accoding to manufacturer's protocol (PCR puification kit, Qiagen)
DNA was eluted 40 µL EB buffer

Digestion of purified PCR-product (P19 with O3 and O4)

Investigator: CR

Aim of the experiment: Digestion of EspP with AgeI and NgoMIV to ligate it in pSB1C3 later on

Procedure: * Batch for preparative digestion:

Volume	Reagent
38,5 µL	PCR product P19 with O3 and O4
1 µL	AgeI
1 µL	NgoMIV
5 µL	CutSmart buffer
4,5	ddH₂O
50 µL	TOTAL

Incubation for 2 h at 37 °C

Sequencing of P26, 27, 28, 30

Investigator: NA

Aim of the experiment:

Sequencing of P26, P27, P28 & P30 (x2)

Procedure:

Sequencing batches were prepared after manufacturer's protocol

(15 μL plasmid DNA (50-100 μM) and 2 μL sequencing primer)

The different plasmids we prepared received the following barcodes:

P26 (VF2) : FR11326643

P27 (VF2) : FR11326642

P28 (VF2) : FR11326641

P30 (VF2) : FR11326640

P30 (VR): FR11326639

Ion exchange chromatography of eGFP

Investigator: NA, JB,JL

Aim of the experiment: purification of eGFP

Procedure:

-use Äkta-Purifier (Q-column for eGFP (quarternary ammonium as stationary phase)). Ask Andy before use!

-Pump A: running buffer (20 mM Tris/HCl pH 8); pump B: elution buffer (20 mM Tris/HCl pH 8 and 1,000 mM (1 M) NaCl

-basic pumpwash before start

-injection in loop

- gradient of ion strength on column.

Results:

Elution of eGFP at ~200 mM NaCl.

[[Image:|top]]

Thursday, May 26^th

Inoculation of cultures with XL-1 F11 and F12 clones

Investigator: JB

Aim of the experiment:

Inoculation of a 5 ml culture with a clone of the previously transformed F11 (Avi-Tag) F12 (Antibody-binding site).

Cloning of ligation F14 +F13

Investigator: MP, EF, JH

Aim of the experiment: new biobrick EspP in pSB1C3

Procedure: * F14 was purified by gelelctrophoresis (1%, 90 V, 40 min)

gelextraction was performed after manufacturer's protocol (Gelextractionkit, Qiagen)
ligation of F14 (EspP) with F13 (dephos. PSB1C3)
transformation according to protocol in competent E.coli XL1 blue

Results:

[[Image:|top]]

Transformation of Biobrick B0034 in XL1-blue

Investigator: JH

Aim of the experiment: Transformation

Procedure:

-10 µl ddH₂O in well of interest (standard distribution kit, plate 4, well 1N)

-1 µl Plasmid (out of well) to cells

-Transformation according to the SOP (incl. rescue)

Transformation of Biobricks K577895 and K577894 in XL1-blue

Investigator: NA

Aim of the experiment: Transformation

Procedure:

-10 µl ddH₂O in well of interest (standard distribution kit, plate 1, wells 14B / 12P)

-1 µl Plasmid (out of well) to cells

-Transformation according to the SOP (incl. rescue)

SDS-PAGE of eGFP-fractions after IEC

Investigator: NA, CG

Procedure: mixing of 80 µl sample with 20 µl SDS buffer and heating at 95°C for 10 min. 1 h staining, 1 night unstaining

Concentrations were measured via nanodrop (A280, extinction coefficient: 55000 M⁻¹cm⁻¹)

Result: concentrations in mg/ml* 1. Lane: 8 µl Marker

2. Lane: 10 µl of fraction 10; c= 0
3. Lane: 10 µl of fraction 11; c= 0,15
4. Lane: 10 µl of fraction 12; c= 0,77
5. Lane: 10 µl of fraction 13; c= 0,29
6. Lane: 10 µl of fraction 14; c= 0,4
7. Lane: 10 µl of fraction 15; c= 0,144
8. Lane: 10 µl of fraction 16; c= 0,085

eGFP is clearly detectable at about 27 kDa. The most pure fractions 12 and 13 were pooled for later biotinylation.

[[Image:|top]]

Friday, May 27^th

Inoculation of pre-cultures with B0034, K577894, KK577895, F13+F14

Investigator: JH

Procedure: * Add 50 µL ampicillin(B0034)/chloramphenicol(K5777894, K577895, F13+F14) to 50 mL LB-medium

Picking colonies from plate (XL1 blue; all rescue)
Incubate over night at 37°C

Inoculation of BL21 (pASK75 (SA1)) culture in 2 L TB-Medium and induction of streptavidin production by addition of tetracycline

Investigator: NA

Aim of the experiment: Production of streptavidin

Procedure: * Ampicillin (2 mL) was added to the Medium (1:1000)

The pre-culture (50 mL) was poured into the Medium
Culture was incubated at 37°C and 140 rpm until OD₅₅₀ reached 0.5
To induce streptavidin expression anhydro-tetrazycline (200 µL) was added to the culture (1:10000)
The culture was incubated at 37°C and 140 rpm for 4 hours

Result:* Streptavidin expression by BL21

Sequencing of P30 (again)

Investigator: NA

Aim of the experiment:

Purification and sequencing of P30

Procedure:* P30 was heated up to 100 °C and centrifugated afterwards to remove proteins from Promotor (if there are some), the supernatant is sequenced

Sequencing batches were prepared after manufacturer's protocol

(15 μL plasmid DNA (50-100 μM) and 2 μL sequencing primer)

The different plasmids we prepared received the following barcodes:

P30 (VF2) : FR11326638

Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium

Investigator: JH, NA

Aim of the experiment: Recombinant expression and purification of Streptavidin

Procedure: * After expression, cultures were transferred into centrifuge tubes and spun down in the centrifuge (4°C, 5000 rpm, 20 mins, F 4X1L rotor)

The supernatant was cast away and the pellet was stored at –20 °C

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: CG

Aim of the experiment: MiniPrep of cloned pSB1C3-A3C5 and –AviTag Plasmids

Procedure: * MiniPrep of inoculated precultures was performed according to manufacturer’s protocol (QIAprep MiniPrep, Qiagen)

Concentrations were measured (in ng/µL):

Colony 1 of pSB1C3- AviTag P32: 120

Colony 2 of pSB1C3- AviTag P33: 110

Colony 1 of pSB1C3-A3C5 P34: 126

Colony 2 of pSB1C3-A3C5 P35: 188

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: NA, JH, CG

Aim of the experiment: Initial vector digestion was performed with NgoMiV and AgeI, producing complementary overhangs. Religated pSB1C3 (due to incomplete vector dephosphorylation) loses the AgeI recognition sequence. Therefore plasmids without insert should run in the supercoiled position at about 1.5 kb, plasmids with inserts should run in the linearised position at 3 kb. Single enzyme digestion was chosen, because the insert is too small to be solved on a gel.

Procedure: * analytic digestion: 3 µl of plasmid DNA P32 to P35 (about 300 ng) were digested with 0.3 µl AgeI-HF in 1 µl CutSmart Buffer and 5.7 µl H2O. The reaction was incubated for 2 h at 37°C.

5 µl digestion mix were loaded on a 1% agarose gel for electrophoresis, 1 kb ladder

Result:

P32 to P35 might carry the insert because they all appear in the linearized position at 3 kb. Therefore plasmid sequencing should confirm the result.

Lane 1: 1 kb ladder

Lane 3: P32

Lane 4: P33

Lane 5: P34

Lane 6: P35

[[Image:|top]]

Monday, May 30^th

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: CR

Aim of the experiment:

Sequencing of P32 and P35

Procedure:

Sequencing batches were prepared according to manufacturers protocol (Mix2Seq Kit, Eurofins Genomics): 15 μL plasmid DNA (50-100 μM) and 2 μL sequencing primer (10 µM)

The plasmids received the following barcodes:

P32 (VF2) : FR11326637

P35 (VF2) : FR11326636

Religation of F13 and transformation in XL1 blue

Investigator: CR

Aim of the experiment:

Re-ligate digested and dephosphorylated pSB1C3 and transformation in XL1blue

Procedure:* Plasmid was ligated with the T4 DNA Ligase following the manufacturer’s protocol (NEB)

52 ng Vector DNA were used
The Ligation mix was incubated for 30 min at RT
7 μL Ligation mixed were used for the transformation of 50 μL XL1 blue.
Transformation was performed following the SOP

Result:* 5 clones on plate

Inoculation of pre-cultures with B0034, K577894, KK577895, F13+F14

Investigator: CR

Procedure: * Add 50 µL ampicillin(B0034)/chloramphenicol(K5777894, K577895, F13+F14) to 50 mL LB-medium

Picking colonies from plate (XL1 blue; all rescue)
Incubate over night at 37°C

Tuesday, May 31^st

Dialysis of eGFP

Investigator: NA

Aim of the experiment: Changing the buffer of eGFP for biotinylation (from TRIS to PBS)

Procedure: * eGFP was poured in a dialysis hose (cut-off 14 kDa)

The hose was then placed in ice cold PBS pH 7.4
The dialysis took place at 4°C overnight

Expression and harvest of Streptavidin (pASK75 (SA1)) in BL21 in TB-medium

Investigator: JB, NA

Aim of the experiment: Recombinant expression and purification of Streptavidin

Procedure: * The pellet was transferred into a beaker of sufficient size and resuspended in fridge-cooled Tris Buffer B (50 mM Tris/HCl (pH = 8.0), 1 mM EDTA)

The solution was homogenized in the PANDA (see SOP)
The resulting lysate was transferred into centrifuge tubes and spun down (4°C, 18,000 rpm, 30 mins, SS34-rotor). The supernatant was cast away and the pellet was resuspended in 6M Gua-HCl (pH = 1.5) at 4°C overnight.

Transformation of P30

Investigator: NA

Aim of the experiment: expression of tet-repressed GFP in E. Coli BL21

Procedure: transformation in competent E. Coli BL21 according to protocol

Result: plates (LB Cam) in incubator for further processing (37 °C)

Cloning of A3C5 and Avi-Tag into pSB1C3

Investigator: CR, JH

Aim of the experiment:

Preculture of pSB1C3 (F13+F14) was red

See if RFP-generator is still inside the pSB1C3

See if ligation of EspP was successful

Procedure: * MiniPrep of P39 was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

pSB1C3 was digested with AgeI and NgOMIV (1.5 µL P39 + 1.5 µL AgeI + 1.5 µL NgoMIV + 3 µL CutSmart + 22.5 µL ddH₂O; incubation for 2h, 37°C)
FastAP was added and the reaction was incubated for another 10min at 37°C
FastAP was inactivated by heating the mix to 75°C for 10min.
The digestion was purified by gelelctrophoresis (1%, 75V, 1h)

Results:

[[Image:|top]]

Unknown signals.
Repeat transformation, digestion, dephosphorylation and ligation of pSB1C3

Re-transformation of P4 (pSB1C3 (RFP-generator)) in XL1 blue

Investigator: JH, CR

Aim of the experiment: Transformation

Procedure: * 2 μL P4 were used for transformation of XL1 blue

Transformation was performed according to SOP (incl. rescue plate)

Competent E. coli XL1 cells

Investigator: JB

Aim of the experiment: Generation of competent E.coli XL1 cells

Procedure: * 5 ml LB w.o. antibiotics (sterile)

Inoculation of one colony of E.coli XL1 cells
Incubate overnight at 37°C

Wednesday, June 1^st

eGFP Biotinylation

Investigator: CG

Aim of the experiment: Biotinylation of dialysed eGFP past IEC

Procedure: * The average concentration of the pooled factions 12 and 13 should be about 0.5 mg/ml (=19 µM)

10 ml of a 100 mM Biotin stock-solution was prepared (340 mg)
3.5 ml of eGFP were biotinylated with 12 µl 100 µM Biotin-solution (20x molar excess)
Incubation at 4 °C overnight

pSB1C3 vector backbone preperation

Investigator: CG

Aim of the experiment: Inoculation of two colonies pSB1C3-RFP Generator

Procedure: * 2x 5 ml LB+Cam medium (1:1000)

Picking colonies from plate (XL1 blue, rescue)
Incubate overnight at 37°C

Generation of GFP expressing E. coli cells

Investigator: CG

Aim of the experiment: Inoculation of BL21 transformed cell with P30

Procedure: * 50 ml LB+Cam medium (1:1000)

Picking colonies from plate (BL21, rescue)
Incubate over night at 37°C

Competent E. coli XL1 cells

Investigator: JB

Aim of the experiment: Generation of competent E. coli XL1 cells

Procedure: * Inoculated 500 µl preculture in 50 ml LB media w.o. antibiotics

Incubated and shaken at 37°C until the OD550 reached 0.44
Transferred into a falcon tube and incubated on ice for 10 mins
Spun down at 3000 g for 10 mins at 4°C, supernatant cast away
Pellet resuspended in 40 ml fridge-cooled MgCl2 (100 mM), once again spun down at 4°C for 10 mins at 3000 g
Supernatant cast away, pellet resuspended in 20 ml cold CaCl2 (50 mM)
Incubation on ice for 30 mins
Centrifugation repeated, supernatant cast away
Pellet resuspended in 2 ml of CaCl2 (50 mM) and glycerol (15%)-solution, aliquoted (50 µl each), frozen in liquid nitrogen and stored at -80°C for further use

Cloning of Pcat-RBS-construct in pSB1A2

Investigator: CG, JH, CR

Aim of the experiment: Pcat (I14033) was cut out of P27 and ligated into P38 for generating the Pcat-RBS (B0034) construct.

Procedure: * P27 was digested with EcoRI and SpeI (4 µL P27 + 1.5 µL EcoRI + 1.5 µL SpeI + 3 µL CutSmart + 20 µL ddH₂O; incubation for 2h, 37°C)

P38 was digested with EcoRI and XbaI (10 µL P27 + 1.5 µL EcoRI + 1.5 µL XbaI + 3 µL CutSmart + 14 µL ddH₂O; incubation for 2h, 37°C)
Preperative gel electrophoresis was performed
For P27 the signal at about 50 bp was cut out and gel extracted according to manufacturer's protocol (Gelextractionkit, Qiagen): 5.9 ng/µl
For P30 the signal at about 2.1 kb was cut out and gel extracted according to manufacturer's protocol (Gelextractionkit, Qiagen): 8.9 ng/µl
Ligation according to SOP

Purification of streptavidin

Investigator: JB

Aim of the experiment: Refolding denaturized streptavidin

Procedure:

- the GdmCl-solved streptavidin was spun down using an SS-34 rotor (~60 min, 18,000 rpm, 4°C)

- The supernatant containing the solved and denaturized protein was transferred into a falcon tube, the pellet cast away

- The protein solution was slowly dripped into 4 l of cooled 1x PBS (in the 4°C room) using a hydraulic pump and stirred overnight

Thursday, June 2^nd

Preparation of pSB1C3 for further use

Investigator: CR

Aim of the experiment: Digestion of pSB1C3 to remove RFP-generator

Procedure: * MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

P40 was digested with AgeI and NgOMIV (2.5 µL P40 + 1.5 µL AgeI + 1.5 µL NgoMIV + 3 µL CutSmart + 21.5 µL ddH₂O)
Incubation over night at 37°C

Generation of GFP expressing E. coli cells

Investigator: CG, JH

Aim of the experiment: Various inductor concentrations

Procedure: * 12x 5 ml LB+Cam media were inoculated with 50 µl preculture

when cultures reached OD550 of 0.5, they were induced with tetrazycline-anhydride of various concentrations (0 ng/µl, 20 ng/µl, 40 ng/µl etc., 200 ng/µl)
t=0, =30, =90, =150, =210, =270 a 100 µl aliquot of each culture was stored at 4 °C in the refrigerator

Dialysis of biotinylated eGFP

Investigator: JH, JB

Aim of the experiment: Purification of biotinylated eGFP

Procedure: * eGFP was poured in a dialysis hose (cut-off 14 kDa)

The hose was then placed in 2 L ice cold Tris/HCl 20 mM pH 8.0
The dialysis took place at 4°C over night

Ligation of F17.1 and F18 and transformation in XL1 blue

Investigator: JH

Aim of the experiment:

Ligation and transformation in XL1blue

Procedure:* Plasmid was ligated with the T4 DNA Ligase following the manufacturer’s protocol (NEB)

9 µL F18 (B0034 in pSB1A2; RBS) and 1 µL of F17.1 (I14033; P(cat)) were used [AmpR]
The Ligation mix was incubated for at least 2h at RT
7 μL Ligation mixed were used for the transformation of 50 μL XL1 blue
Transformation was performed following the SOP [AmpR], incl. rescue plate

Friday, June 3^rd

Cloning of F8, F9 and F14 into F20

Investigator: CR

Aim of the experiment: Cloning of A3C5, Avi-Tag and into pSB1C3

Procedure: * FastAP was added and the digestion of F20 and was incubated for another 15 min at 37°C

The digestion was purified by gelelctrophoresis (1%, 75V, 1h)
Plasmid backbone (signal 1) was cut from the gel
Gelextraction was performed after manufacturer's protocol (Gelextractionkit, Qiagen)

Results:

[[Image:|top]]* Signal 1: F20

[F20]= 13,8 ng/μL

Generation of GFP expressing E. coli cells

Investigator: CG, NA

Aim of the experiment: SDS PAGE for verification of successful GFP expression

Procedure: * SDS electophoresis according to SOP

Lane 1: #12, 90 min
Lane 2: #12, 150 min
Lane 3: #12, 210 min
Lane 4: #12, 270 min
Lane 5: 8 µl Ladder

Result:

[[Image:|top]]

Inoculation of colonies from F19

Investigator: CG

Aim of the experiment: three colonies were picked from yesterdays transformation

Procedure: * 3x 5 ml LB+Amp media

Each culture was inoculated with one colony
Incubation at 37°C overnight

Cloning of F21, F22 and F23 into F20

Investigator: CG

Aim of the experiment: Ligation of F21, F22 and F23 into F20 and transformation

Procedure: * Ligation was performed according to the SOP with: 0.7 µl F8 (1:10) / 9.3 µl F20, 0.7 µl F9 (1:10) / 9.3 µl F20, 9.1 µl F14 / 0.9 µl F20

Transformation was performed according to the SOP on LB Cam-Agar

Precipitation of Streptavidin

Investigator: NA, JB, LK

Aim of the Experiment: Purification of Streptavidin

Procedure:* Slowly add (NH⁴⁺)₂SO₄^2- until the concentration reaches 40% (23,1 g/100mL) while stirring

Let it stir over night to ensure complete precipitation
Centrifuge at 8500 rpm at 4°C for 15min (rotor: SLA1500), dismiss the pellet and use the supernatant for further processing
Slowly add (NH⁴⁺)₂SO₄^2- until the concentration reaches 70% (19,1 g/100mL) while stirring
Let it stir over night (slowly to avoid foaming)
Centrifuge at 8500 rpm at 4°C for 15min (rotor: SLA1500), dismiss the supernatant and use the pellet for further processing
Resuspend the protein pellet in a minimum volume of TRIS/HCl; pH 8; 20mM (NO SALT) (6 mL per pellet), the pellets where then united and titrated with Buffer until the solution became clear. Total amount of buffer: 40mL
Transfer to 50 mL Falcon Tubes and centrifuge at full speed for 15 min. Filter the supernatant through 45nm sterile filter and keep the pellet (rescue).

Saturday, June 4^th

Inoculation of colonies from F21, F22, F23

Investigator: NA

Aim of the experiment: three colonies were picked from each transformation

Procedure: * 3x 5 ml LB+Cam media for each fragment

Each culture was inoculated with one colony
Incubation at 37°C overnight

MiniPrep of F19

Investigator: NA

Aim of the experiment: Extraction of Ligation: F17+F18 (starke RBS+P(cat))

Procedure:

MiniPrep was performed after manufacturer's protocol (QIAprep MiniPrep, Qiagen)

Concentrations: K1= 66,2 ng/µl; K2= 60,2 ng/µl; K3= 104,4 ng/µl

Monday, June 6^th

Cloning of TetR from p28 into p31

Investigator: LK, JH, NA

Aim of the experiment: Cloning TetR in front of weak RBS in pSB1C3

Procedure: * Digestion of p28 with EcoR1 and Spe1 (20µl DNA, 2,5 µl each enzyme, 5µl CutSmart buffer, 20µl ddH₂O) and p31 (4µl DNA, 1µl EcoRI / XbaI, 2µl, 12µl DNA) -> Incubation at 37°C for 2h.

Electrophoresis: 1% Agar, 15 min , 70 V (oben p31 und p41, unten p28)

[[Image:|top]]

* Gelextraction  concentrations: p28 (F25) = 2,3 ng/µl

p31 (F24) = 7,8 ng/µl

* Ligation with 6,3 µl vector (F24) and 1,7 µl Insert (F25) + 1µl Buffer + 1µl Ligase  over night, 16°C

SDS-PAGE of Streptavidin

Investigator: NA, JH, LK

Procedure: mixing of 80 µl sample with 20 µl SDS buffer and heating at 95°C for 10 min. 1 h staining, 1 night unstaining. Concentration of streptavidin was measured via UV/VIS spectroscopy.

<math>c=\frac{A}{ℇ\ast d}\ast M\ast \mathit{Verd}.\rightarrow </math> c = 23,5 mg/ml

Result: * 1. Lane: 8 µl Marker

2. Lane: 5 µl of Streptavidin_1:10
3. Lane: 5 µl of Streptavidin_1:50
4. Lane: 5 µl of Pellet_1:500
5. Lane: 5 µl of after 40% precipitation_1:10
6. Lane: 5 µl of supernatant after 70% precipitation and centrifugation

Successful Streptavidin production verified.

[[Image:|top]]

Sequencing of P41

Investigator: NA

Aim of the experiment: Sequencing of P41

Procedure:

Sequencing batches were prepared after manufacturer's protocol (15 µL plasmid DNA (100 ng/ µl) and 2 µL sequencing primer (VF2))

The plasmids prepared received the following barcode:* P41 : FR11326633