Team:Bielefeld-CeBiTec/Notebook/Labjournal

Labjournal

2017-04-03 - 2017-04-09

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: electroporation transformation of BBa_K1416000

Procedure:

standard electroporation protocol

used electro competend DH5alpha from NEB
streaked out on LB-plates with Cam

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: electroporation transformation of BBa_KJ36848

Procedure:

standard electroporation protocol

used electro competend DH5alpha from NEB
streaked out on LB-plates with Cam

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of transformed BBa_K1416000

Procedure:

standard procedure

1,25µl Cam in 5mL LB

2017-04-10 - 2017-04-16

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K1416000

Procedure:

standard protocol

517,9 ng/µL

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of transformed BBa_J36848

Procedure:

standard procedure

1,25µl Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_J36848

Procedure:

standard protocol

231,8 ng/µL

2017-05-29 - 2017-06-04

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of BBa_E0400

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with Amp

2017-06-05 - 2017-06-11

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of transformed BBa_E0400

Procedure:

standard procedure

5,0µl Amp in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_E0400

Procedure:

standard protocol

88,4 ng/µL

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of BBa_K525998

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with Cam

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of transformed BBa_K525998

Procedure:

standard procedure

1,25µl Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K525998

Procedure:

standard protocol

140,9 ng/µL

2017-06-12 - 2017-06-18

Assemble the fragments to get the Part BBa_K2201220

Investigators: Yannic Kerkhoff

Superior experiment: Gibson assembly of F1,F3,F5

Procedure:

standard Gibson Assembly protocol

Cellgrowth with the barnaseplasmid

Investigators: Olga Schmidt, Denise Kerkhoff, Christina Drake

Superior experiment: Overnightculture of barnase

Procedure:

3 ml LB-medium and 0,75 µl chloramphenicol
inkubation overnight 37°C, 140rpm

To get and purified the plasmid

Investigators: Christina Drake

Superior experiment: Plasmidisolation of barnase-plasmid

Procedure:

protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
concentration measurement by nanoprop
sequenzingorder

Duplicate the parts

Investigators: Christina Drake, Denise Kerkhoff

Superior experiment: Transformation via Distribution of the parts BBA_J04450, BBa_I746909, BBa_K80800 and BBa_psB1K5

Procedure:

transformation via electroporation
plated out: BBa_J04450 on tetracycline, BBa_I74909 and BBa_K808000 on chloramphenicol, BBa_psB1K5 on kanamycin

Duplicate the part

Investigators: Christina Drake, Denise Kerkhoff

Superior experiment: Transformation of BBa_psB6A1

Procedure:

transformation via electroporation
plated out on amphenicol

Cellgrowth with the plasmids

Investigators: Denise Kerkhoff, Christina Drake

Superior experiment: Overnightculture of BBA_J04450, BBa_I746909 and BBa_K808000

Procedure:

3 ml LB-medium and at BBA_J04450 3µl tetracycline and atBBa_I746909 and BBa_K808000 0,75 µl chloramphenicol
inkubation overnight 37°C, 140rpm

To get and purified the plasmid

Investigators: Christina Drake

Superior experiment: Plasmidisolation of BBA_J04450, BBa_I746909 and BBa_K80800-plasmids

Procedure:

protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
concentration measurement by nanoprop:

BBA_J04450 26ng/µl and 25,7ng/µl
BBa_I746909 90,2ng/µl
104,3 ng/µl -BBa_K808000 118,3 ng/µl

and 146,6 ng/µl
sequenzingorder

2017-06-19 - 2017-06-25

Cellgrowth with the plasmid

Investigators: Denise Kerkhoff

Superior experiment: Overnightculture of BBA_psB3K5

Procedure:

3 ml LB-medium and 2,5 µl kanamycin
inkubation overnight 37°C, 140rpm

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of assembled fragments F5,F2,F4

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with Cam

multiplication, verification and purification of the needed fragment F2: GFPLinker2

Investigators: Yannic Kerkhoff

Superior experiment: Q5-PCR, gel and PCR-cleanUp of F2

Procedure:

standard Q5-PCR protocol

template: BBa_E0400
primer fwd: 17gk
primer rev: 17gn
annealing temperature: 58°C
extension time: 50 seconds

standard PCR-cleanUp protocol

125,8 ng/µL

multiplication, verification and purification of the needed fragment F4: StrepLinker2

Investigators: Yannic Kerkhoff

Superior experiment: Q5-PCR, gel and PCR-cleanUp of F4

Procedure:

standard Q5-PCR protocol

template: BBa_KJ36848
primer fwd: 17gp
primer rev: 17gq
annealing temperature: 61°C
extension time: 30 seconds

standard PCR-cleanUp protocol

127,0 ng/µL

multiplication, verification and purification of the needed fragment F5: linearized pSB1C3

Investigators: Yannic Kerkhoff

Superior experiment: Q5-PCR, gel and PCR-cleanUp of F5

Procedure:

standard Q5-PCR protocol

template: K1416000
primer fwd: 17gj
primer rev: 17gl
annealing temperature: 59°C
extension time: 120 seconds

standard PCR-cleanUp protocol

258,4 ng/µL

Assemble the fragments to get the Part BBa_K2201221

Investigators: Yannic Kerkhoff

Superior experiment: Gibson assembly of F2,F4,F5

Procedure:

standard Gibson Assembly protocol

Screening for positive transformations containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: GoTaq PCR of 7 clones of BBa_K2201221

Procedure:

standard GoTaq-protocol

template: colonie pcr clones of GA_K2201221
primer fwd: Präfix_fwd
primer rev: Suffix_rev
annealing temperature: 56°C
extension time: 40 seconds

positive clone 2

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of the positive clone 2 of BBa_K2201221

Procedure:

standard procedure

1,25µl Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K2201200 clone 20

Procedure:

standard protocol

240,6 ng/µL

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K2201221 clone 2

Procedure:

standard protocol

208,8 ng/µL

2017-06-26 - 2017-07-02

Check the sequence

Investigators: Christina Drake

Superior experiment: Sequenzingorder barnase

Procedure:

concentration measurement by nanoprop: 177 ng/µl
result: sequence was not barnase

Plasmidisolation and gibson assembly of pSB1C3-PlacUV5_PtNTT2 c30

Investigators: Camilla Maerz

Superior experiment: Construction of pSB1C3-PlacUV5_PtNTT2

Procedure:

Plasmid DNA purification kit

Nucleo spin plasmid protocol

Gibson Assembly protocol
transformation via heat shock protocol in E. coli DH5alpha

Colony PCR of T7RNAP from E. coli KRX

Investigators: Olga Schmidt

Superior experiment: Construction of the positive selection plasmid

Procedure:

PCR with Grimson Taq polymerase (per reaction, 25 µL):

5 µL 5x Buffer
2.5 µL dNTPs (2 mM)
0.5 µL 3'-Primer (10 mM)
0.5 µL 5'-Primer (10 mM)
0.125 µL Grimson Taq polymerase
1 picked bacterial colony as template
ad 25 µL H20

Primer annealing: 64 °C
Elongation: 150 s
Agarose gel electrophoresis (1%)

100 V, 30 min

Isolate T7RNAP from the chromosome of KRX-e.coli

Investigators: Christina Drake

Superior experiment: PCR of T7RNAP from KRX-e.coli

Procedure:

Q5 High-Fidelity PCR

annealingtemparatur 64°C, elogationtime 1 min

1%-agarose-TAE-gel

1kb Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye

Plated out Barnase from strain collection

Investigators: Christina Drake

Superior experiment: Plated out Barnase from strain collection

Procedure:

solved DNA in 100 µl SOC-medium
plated out on chloramphenicolplate
overnightincubation at 37°C

Colony PCR of pK18mobsacB-del_codA

Investigators: Camilla Maerz

Superior experiment: Construction of pK18mobsacB_codA_del

Procedure:

Go Tag (Promega) protocol

Primer: VR, VF2
Primer annealing: 56 °C
Elongation: 140 s

PCR of pSB1C3

Investigators: Olga Schmidt

Superior experiment: General

Procedure:

PCR

Q5 High-fidelily (NEB) protocol
Denaturation: 64 °C
Elongation: 60 s

Agarose gel electrophoresis (1%)

100 V, 30 min

Gibson assembly of T7RNAP in pSB1C3

Investigators: Olga Schmidt

Superior experiment: Construction of the selection plasmid

Procedure:

Gibson Assembly protocol
transformation via heat shock protocol in E. coli BL21

Colony PCR of pSB1C3_T7RNAP c1-8

Investigators: Olga Schmidt

Superior experiment: Construction of the selection plasmid

Procedure:

PCR

Q5 High-fidelily (NEB) protocol
Denaturation: 64 °C
Elongation: 80 s

Agarose gel electrophoresis (1%)

100 V, 30 min

Colony PCR of barnase (26.06.2017) c1&2

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

PCR

Go Tag (Promega) protocol
Danaturation: 58 °C
Elongation: 35 s

Agarose gel electrophoresis (1%)

100 V, 30 min

2017-07-03 - 2017-07-09

Check the correct barnasegen

Investigators: Christina Drake

Superior experiment: PCR of barnase

Procedure:

Go-Taq-protocol

annealingtemparatur 58°C, elogationtime 30 s

1%-agarose-TAE-gel

100bp Marker Geneuler and 5 µl sample with 1 µl 6*Loading Dye
result: PCR-product could not be barnase

2017-07-10 - 2017-07-16

Biobrick Assembly of 2B1

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

BioBrick Assembly

RuBisCoTAG mRFP as upstream part, T7-Terminator as downstream part, pSB1A3 as destination plasmid

transformation via heat shock and DH5α Cells

used 5µl from the ligation

plated out on Amp plates and let them grow over night

Check the sequence

Investigators: Christina Drake

Superior experiment: Sequenzingorder barnase

Procedure:

result: sequence was not barnase

>16.07.2017

tRNA

Overnightculture of TyrRS-Heidelberg-plasmid

CDR

Cellgrowth with the TyrRS-Heidelberg-plasmid

3 ml LB-medium and 0,75 µl chloramphenicol
inkubation overnight 37°C, 140rpm

Transformation of T7-Promotor

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

transformation via electroporation of cells with T7-Promotor

Plasmidisolation of TyrRS-Heidelberg plasmid

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

Plasmid DNA purification kit (Macherey-Nagel)

Nucleo spin plasmid protocol

Preculture of colonies with T7-Promotor

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

used 5µl LB media and kanamycin

2017-07-17 - 2017-07-23

Isolate the tRNA

Investigators: Christina Drake

Superior experiment: PCR of TyrRS-Heidelberg-plasmid

Procedure:

plasmidisolation with MN protocol 5
Q5 High-Fidelity PCR

annealingtemparatur 53°C, elogationtime 20s

Gibbsonassembly with 5µl backbone and 0.5 µl insert
transformation via heat shock
overnight incubation at 37°C

Isolation of T7-Promotor

Investigators: Laura Schlueter

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

Nucleospin Plasmidisolation protocol

got four products with the concentrations 50.1 ng/µl, 16.3 ng/µl, 24,2 ng/µl and 2.1 ng/µl

Colony PCR with 2B1 Colonies

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

GoTaq® G2 PCR

Biobrickassembly of 2B2 to 2B8, B1 and A1

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

BioBrick Assembly

digest and ligate RuBisCo parts as upstreampart and T7-Terminator/Promotor as downstreampart and pSB1A3 as destination plasmid

ligate TAG2 and T7-Terminator to 2B2
ligate TAG111 and T7-Terminator to 2B3
ligate TAG474 and T7-Terminator to 2B4
ligate TAG2+TAG111 and T7-Terminator to 2B5
ligate TAG2+TAG474 and T7-Terminator to 2B6
ligate TAG111+TAG474 and T7-Terminator to 2B7
ligate TAG2+TAG111+TAG474 and T7-Terminator to 2B8
ligate RuBisCo mRFP and T7-Terminator to B1
ligate Carboxysome and T7-Promotor to A1

preculture of positive 2B1 colony

Transformation of test devices

Investigators: Camilla Maerz

Superior experiment: Interlab study

Procedure:

dilute test devives (distribution plate 6 & 7) in 10 µL ddH20
Transformation

1 µL plasmid from each plate

transformation via heat shock protocol in E. coli XL1-blue

Transformation of 2B2-2B8, B1 and A1

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

transformation via heat shock

Plasmidisolation of 2B1

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

Nucleospin Plasmidisolation protocol

got 47 ng/µl and 41 ng/µl

Reverse the insert

Investigators: Christina Drake

Superior experiment: PCR of T7GFP

Procedure:

Q5 High-Fidelity PCR

Insert: annealingtemparatur 60°C,
Backbone:elogationtime 30s: annealingtemparatur 60°C, elogationtime 1 min
1%-agarose-TAE-gel

result: PCR didn`t work

Reverse the insert

Investigators: Christina Drake

Superior experiment: PCR of T7GFP

Procedure:

1%-agarose-TAE-gel
result: PCR did work
Gibson Assembly
transformation via heat shock

PCR of BBa_I746909 and pSB1C3

Investigators: Olga Schmidt

Superior experiment: Integration of CDS of BBa_I746909 in pSB1C3

Procedure:

PCR of I746909

Q5 High-fidelily (NEB) protocol
Primer: 17lk & 17lj
Denaturation: 60 °C
Elongation: 40 s

PCR of pSB1C3

Q5 High-fidelily (NEB) protocol
Primer: 17ll & 17li
Denaturation: 60 °C
Elongation: 70 s

Agarose gel electrophoresis (1%)

100 V, 30 min

Preparation of over-night culture

Investigators: Camilla Maerz

Superior experiment: Interlab study

Procedure:

Over-night culture of negative control, positive control, test device 1-6

LB-media + Cm were inoculated
Cultures were incubated over night (37°C, 200 rpm)

2017-07-24 - 2017-07-30

Plasmidisolation of pSB1C3_I746909

Investigators: Olga Schmidt

Superior experiment: Integration of CDS of BBa_I746909 in pSB1C3

Procedure:

Plasmid DNA purification kit (Macherey-Nagel)

Nucleo spin plasmid protocol

Repeat the degestion and transformation from the 18th and 19th july

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

Cellgrowth with the plasmid

Investigators: Christina Drake

Superior experiment: Overnightculture of tRNA-psB1C3

Procedure:

3 ml LB-medium and 0,75 µl chloramphenicol
inkubation overnight 37°C, 140rpm

Cloning of the ori (pSB6A1) in pSB1C3

Investigators: Laura Schlueter

Superior experiment: aaRS Plasmid (without aaRS)

Procedure:

Gibson Assembly Master Mix

Backbone: 3.28 µL Ori 1.0: 1.73 µL
Backbone: 0.32 µL Ori 1.1: 4.68 µL
Backbone: 0.90 µL Ori 3.0: 4.10 µL

Trafo: heatshock
not successfull because of the wrong overlapps (try again)

Colony PCR of the Retrafo of pRS (because of mixed culture)

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Sequencing was not succesfull (maybe mixed colony), so I did a retrafo with the aim of a successful sequencing (incorporation of the aaRS in pSB1C3)

pRS 3: 1-5
pRS 4: 1-5
pRS 5: 1-5
pRS 7: 1-5
no positive result (no band at 1000 bp)

Amplification of pSB1C3 for the cloning of the ori in pSB1C3

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

PCR with Q5 High-Fidelity Polymerase Master Mix, Primer jb,jn, 65°C
gelelectrophoresis, 1% Agarose
band 2000-2500 bp (supposed to be around 2200 bp)
Clean up from the gel

pSB1C3(nur ori): 22.2 ng/µL

Amplification of pSB1C3 fragments for a 4 part Gibson (see page 4 in the paper-labbook)

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

PCR, Q5 Master Mix

Primer jj, jn for fragment 2, 65°C
Primer jb, ht for fragment 4, 65°C

gelelectrophoresis, 1% Agarose

band at 300 (fragment 2, supposed to be around 326 bp)
band at 1200 (fragment 4, supposed to be around 1233 bp)

Clean up from the gel

fragment 2: 18.5 ng/µL (overlapp(aaRS)_pSB1C3_overlapp(ori))
fragment 4: 19.1 ng/µL (overlapp(ori)_pSB1C3_overlapp(aaRS))

Colony PCR of 2B2-2B8, B1 and A1

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

GoTaq® G2 PCR
most have negative results -> create new destination plasmid pSB1A3

Colony PCR of retrafo and more colonies of the pRS

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

GoTaq Master Mix, Primer vr,vf

2 strong bands at 400 bp and 600 bp, small bands of pRS3 6 and pRS4 9 at around 1200-1500 bp
bands of pRS31, pRS32, pRS34, pRS36, pRS39 at around 1200-1500 bp

GoTaq Master Mix, Primer hq, jk (Gibson Assembly primer) with the colonies pRS31, pRS32, pRS34, pRS36, pRS39

pRS31, pRS32, pRS39 : strong bands at around 1200 bp

the two positive colony PCRs (vf-vr, hq-jk) for these probes, successful integration of the Tyr-aaRS in pSB1C3 seems to be successfull
pRS31, pRS32, pRS39 send to sequencing

pRS31, pRS32, pRS39 plasmid isolation

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Macherey-Nagel purification kit

pRS31: 324.1 ng/µL
pRS32: 231.8 ng/µL
pRS39: 225.5 ng/µL

send tosequencing
pRS31, pRS39 have the pSB1C3 with the Tyr-aaRS incorporated (but on position 32, the Arginine (cgt) should be changed to an Leucine (ctg), but it is not. On position 290 there really is a missing Leu, on position 268 the Asparagin is changed to an Arginine)

2017-07-31 - 2017-08-06

construct a biobrick

Investigators: Christina Drake

Superior experiment: Aquacloning of tRNA and backbone

Procedure:

transformation via heatschock

plasmid isolation of the positive colonies of the retrafo pRS4 4 (wrong!) and pRS3 12

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Macherey-Nagel purification kit with each 3 mL culture

pRS4 4: 44 ng/µL
pRS3 12: 68.3 ng/µL
not send to sequencing jet

4 part Gibson and Trafo

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix with 5 µl template

pSB1C3 fragment (2): 18.5 ng/µL around 350 bp 2.2 µL
pSB1C3 fragment (4): 19.1 ng/µL around 1300 bp 0.8 µL
oK1.1 (pMB1 from pSB6A1): 5.8 ng/µL around 1300 bp 1.8 µL
Tyr-aaRS: 68.5 ng/µL around 1000 bp 0.2 µL

Trafo via heatshock

Gibson of the ori in pSB1C3 (pori) and Trafo

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix with 5 µL template

pSB1C3(nur ori): 22.2 ng/µL around 2100 bp 1.57 µL
ok1.0: 5.80 ng/µL around 1258 bp 3.43 µL

Trafo via heatshock

Transfer positive colonies

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

picked and transfered positiv colonies of 2B2, 2B6, 2B7, 2B8 and A1 onto a new plate
incubate over night
2B2 and 2B8 are contaminated with negativ colonies

construct a biobrick

Investigators: Christina Drake

Superior experiment: Aquacloning of tRNA and backbone

Procedure:

transformation via heatschock

construct a biobrick

Investigators: Christina Drake

Superior experiment: Gibbsonassembly of barnase and backbone

Procedure:

transformation via heatschock

Preculture of 2B6, 2B7 and A1

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

used 3µl LB media

Cellgrowth with the plasmid

Investigators: Christina Drake

Superior experiment: Overnightculture of tRNA-psB1C3 and barnase-psB1C3

Procedure:

3 ml LB-medium and 0,75 µl chloramphenicol
inkubation overnight 37°C, 140rpm

Plasmidisolation

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

Nucleospin Plasmidisolation protocol

got 67.3 ng/µl of 2B6
got 32.7 ng/µl of 2B7
got 51.6 ng/µl of A1

To get and purified the plasmid

Investigators: Christina Drake

Superior experiment: Plasmidisolation of barnase-plasmid and tRNA-plasmids

Procedure:

protocol from Macherley-Nagel: Plasmid DNA purification, protocol 5: NucleoSpin
concentration measurement by nanoprop
sequenzingorder

Redo digestion of the destination plasmid

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

analyze the destination plasmid digestion via gelelectrophoresis

pSB1A3 digestion was not effective

redo the digestion and analyze it on the gel

new dgestion was very effective

2017-08-07 - 2017-08-13

Amplification of the backbone (pSB1C3) and insert (Tyr-aaRS)

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

PCR Q5 Polymerase (no Master Mix)

for 50 µL reaction

10 µL Reaction Buffer
5 µL 2mM dNTPs
1 µL Template
0,5 µL High Fidelity DNA Polymerase
10.0 µL High GC Enhancer
18.5 µL ddest H2O
5.0 µL Primer

pSB1C3 (from RuBisCo, ncAA team): ht, jk 65°C
aaRS (from Heidelberg) : hq, jk 65°C (mistake! should be 58°C, redone identically with 58°C)

digestion of the PCR products with Dpn1: 5 µL CutSmart Reaktionsbuffer and 1 µL Dpn1 per 50 µL PCR product, on 37°C over night)

agarose-gelelectrophhoresis, 1% (bands of the right size)

No further use, because results of the sequencing which show that the integration of the incorporation of the Tyr-aaRS in pSB1C3 was already successful (30.06.2017)

Ligation of 2B2-2B5 and B1

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

Ligation protocol from the BioBrick Assembly

Variation: incubated 1h at RT instead of 10min

characterisation of T7GFP revers

Investigators: Christina Drake

Superior experiment: preparation of KRX-competent cells

Procedure:

heatschocktransformation of T7GFP and T7GFP revers in KRX

construct a biobrick for the selectionplasmid

Investigators: Christina Drake

Superior experiment: PCR of T7RNA-Polymerase

Procedure:

1%-agarose-TAE-gel
result: PCR did work
Gibson Assembly
transformation via heat shock

Amplification of the backbone (pRS31) and insert (ori from pSB6A1, oK1.0) for Gibson assembly

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

PCR Q5 Polymerase (protocoll of 07.08.2017)

pRS31: primer jb, jn 65°C
oK1: primer jl, jc 65°C

digestion with Dpn1 (protocoll of 07.08.2017)
agarose-gelelectrophhoresis, 1%

band around 1200 bp (oK1.1) / 2500 bp (pRS31), positive

Gibson Assembly and Trafo of the pRS31 and oK1.0

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

agarose-gelelectrophhoresis, 1%
purification from the gel with the Macherey-Nagel purification kit

oK1.1: 22.5 ng/µL
pRS32: 9.5 ng/µL

Gibson Assembly Master Mix with:

4.1 µL of oK1.1: around 2480 bp 9.5 ng/µL
0.9 µL of pRS32: around 1250 bp 22.5 ng/µL

Trafo via heatshock

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of assembled fragments F13 and NPA-RS gene synthesis

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with Cam

multiplication, verification and purification of the needed fragment F13: linearized ONBY-Part with removal of the ONBY-RS

Investigators: Yannic Kerkhoff

Superior experiment: Q5-PCR, gel and PCR-cleanUp of F13

Procedure:

standard Q5-PCR protocol

template: K1416000
primer fwd: 17hl
primer rev: 17mv
annealing temperature: 63°C
extension time: 90 seconds

standard PCR-cleanUp protocol

80,6 ng/µL

Assemble the fragments to get the Part BBa_K2201200

Investigators: Yannic Kerkhoff

Superior experiment: Gibson assembly of F13 and NPA-RS gene synthesis

Procedure:

standard Gibson Assembly protocol

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of assembled fragments F5,F1,F3

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with Cam

multiplication, verification and purification of the needed fragment F1: GFPLinker1

Investigators: Yannic Kerkhoff

Superior experiment: Q5-PCR, gel and PCR-cleanUp of F1

Procedure:

standard Q5-PCR protocol

template: BBa_E0400
primer fwd: 17gk
primer rev: 17gm
annealing temperature: 58°C
extension time: 50 seconds

standard PCR-cleanUp protocol

15,9 ng/µL

multiplication, verification and purification of the needed fragment F3: StrepLinker1

Investigators: Yannic Kerkhoff

Superior experiment: Q5-PCR, gel and PCR-cleanUp of F3

Procedure:

standard Q5-PCR protocol

template: BBa_KJ36848
primer fwd: 17go
primer rev: 17gq
annealing temperature: 61°C
extension time: 30 seconds

standard PCR-cleanUp protocol

17,9 ng/µL

Biobrickassembly

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

BioBrick Assembly
repeat digestion of T7-Terminator downstrampart
ligate over night

Transformation of new plasmids

Investigators: Maximilian Edich

Superior experiment: Biobrick Construction with RuBisCo Parts

Procedure:

transformation via heat shock

multiplication, verification and purification of the needed fragment F15: linear GLS1

Investigators: Yannic Kerkhoff

Superior experiment: Q5-PCR, gel and PCR-cleanUp of F15

Procedure:

standard Q5-PCR protocol

template: P2: GLS2
primer fwd: 17go
primer rev: 17gm
annealing temperature: 60°C
extension time: 190 seconds

standard PCR-cleanUp protocol

36,9 ng/µL

Primer annealing preparation

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

Both Primers (17jh; 17ji) were resuspended in 1x Composite Buffer
Dilutions: 100 µM, 10 µM, 5 µM, 2.5 µM, 0.5 µM
OD 260 were measured to determine the real concentration

2017-08-14 - 2017-08-20

Screening for positive transformations containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: GoTaq PCR of 4 clones of BBa_K2201220

Procedure:

standard GoTaq-protocol

template: colonie pcr clones of GA_K2201220
primer fwd: Präfix_fwd
primer rev: Suffix_rev
annealing temperature: 56°C
extension time: 40 seconds

positive clone 3

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of the positive clone 3 of BBa_K2201220

Procedure:

standard procedure

1,25µl Cam in 5mL LB

Primer annealing of 17jh & 17ji

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

Protocol for annealing oligonucleotides (sigmaaldrich)

Eqimolar Primers were mixed (50 µL)
Thermal profile (thermocycler)

95 °C, 2 min
Cool to 25 °C over 45 min
Cool to 4 °C for temporary storage

Integration of barnase in pSB1C3

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

Restriction Digest of pSB1C3_I749606

5 µL 10x Universal buffer
5.54 µL plasmid
1µL XbaI
1µL PstI
filled up to final volume 50 µL with ddH20
Incubation: 30 min, 37 °C
Heatinactivation: 20 min, 80 °C

Agarose gel electrophoresis (1%)

100 V, 30 min

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

Gibson Assembly protocol
transformation via heat shock protocol in E. coli DH5alpha
transformation via electroporation protocol in E. coli DH5alpha

PCR of T7RNAP_BL

Investigators: Olga Schmidt

Superior experiment: Construction of the selection plasmid

Procedure:

PCR

Go Tag (Promega) protocol
Danaturation: 56 °C
Elongation: 180 s

Agarose gel electrophoresis (1%)

100 V, 30 min

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K2201220 clone 3

Procedure:

standard protocol

108,9 ng/µL

Combination of two biobricks to get the composite part BBa_K2201320 for protein expression

Investigators: Yannic Kerkhoff

Superior experiment: Biobrick Assembly of BBa_K608006 and BBa_K2201220

Procedure:

Standard BioBrick Assembly protocol

Backbone: BBa_J04450, 7,7µL
upstream: BBa_K608006, 3,6µL
downstram: BBa_K2201220, 5,0µL
Ligation time was 12 h at 8 °C

Colony and plasmid PCR of T7RNAP_BL c1-4

Investigators: Olga Schmidt

Superior experiment: Construction of the selection plasmid

Procedure:

PCR

Go Tag (Promega) protocol
Primer: VR & VF
Danaturation: 56 °C
Elongation: 180 s

Agarose gel electrophoresis (1%)

100 V, 30 min

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of the Biobrick Assembly product BBa_K2201320

Procedure:

standard heatshock protocol

used own produced chemo competend BL21(DE3)
streaked out on LB-plates with Cam

Colony PCR of pSB1C3_barnase (gblock) c1-70

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

PCR

Go Tag (Promega) protocol
Primer: Vr & VF2
Danaturation: 56.5 °C
Elongation: 40 s

Agarose gel electrophoresis (1%)

100 V, 30 min

Screening for positive transformations containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: GoTaq PCR of 19 clones of BBa_K2201320

Procedure:

standard GoTaq-protocol

template: colonie pcr clones of GA_K2201320
primer fwd: T7-eva_fwd
primer rev: Suffix_rev
annealing temperature: 51°C
extension time: 80 seconds

positive clone 19

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of the positive clone 19 of BBa_K2201320

Procedure:

standard procedure

1,25µl Cam in 5mL LB

Combination of two biobricks to get the composite part BBa_K2201321 for protein expression

Investigators: Yannic Kerkhoff

Superior experiment: Biobrick Assembly of BBa_K608006 and BBa_K2201221

Procedure:

Standard BioBrick Assembly protocol

Backbone: BBa_J04450, 2,0µL
upstream: BBa_K608006, 3,6µL
downstram: BBa_K2201221, 2,4µL
Ligation time was 12 h at 8°C

Primer annealing of 17jl & 17jm

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

Both Primers were resuspended in ddH20 to a final concentration of 100 µM
HEPES annealing standard protocol
Aqua annealing standard protocol

Final volume: 20 µL

Agarose gel electrophoresis (2%)

60 V, 50 min

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of BL21(DE3) BBa_K2201320

Procedure:

standard procedure

1,25µl Cam in 10mL LB
12 h at 37°C

expression of the fusion protein

Investigators: Yannic Kerkhoff

Superior experiment: preculture of BL21(DE3) BBa_K2201320 with IPTG

Procedure:

standard procedure

1,25µl Cam in 10mL LB
12 h at 37°C
induced with IPTG 0.8mM

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K2201320 clone 19

Procedure:

standard protocol

454,4 ng/µL

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of BL21(DE3) BBa_K2201320

Procedure:

standard procedure

1,25µl Cam in 10mL LB
12 h at 37°C

expression of the fusion protein

Investigators: Yannic Kerkhoff

Superior experiment: preculture of BL21(DE3) BBa_K2201320 with IPTG

Procedure:

standard procedure

1,25µl Cam in 10mL LB
12 h at 37°C

Ligation of pSB1C3 and barnase (gblock)

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

Ligation reaction

7 µL vector
3 µL gblock
2 µL T4 DNA ligase buffer
1 µL T4 DNA ligase
17 µL ddH20

Incubation: RT, 1 h

Colony PCR of pSB1C3_barnaseBL c1-60

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

PCR

Q5 High-fidelily (NEB) protocol
Denaturation: 56.5 °C
Elongation: 45 s

Agarose gel electrophoresis (1%)

100 V, 30 min

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of the Biobrick Assembly product BBa_K2201321

Procedure:

standard heatshock protocol

used own produced chemo competend BL21(DE3)
streaked out on LB-plates with Cam

- standard cultivation protocoll

Investigators: Yannic Kerkhoff

Superior experiment: Cultivation of BL21(DE3) with BBa_K2201320

Procedure:

2x 100mL LB-media with 25µL Cam in 1000mL flasks

innoculation with 3.3 mL preculture of OD 3 to get a starting OD of 0.1
incubation for four hours to a OD of 0.6
addition of 500µL 0.5mM IPTG each
incubation for 8 hours at 37°C and 24 hours at 18°C
harveting of the cells

Native PAGE of annealed primers (17jl & 17jm)

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

Native DNA PAGE standard protocol

tested samples:

HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM

Variation: ~40 mA

2017-08-21 - 2017-08-27

Native PAGE of annealed primers (17jl & 17jm)

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

Native DNA PAGE standard protocol

tested samples:

HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM

Variation: 100 V, 1 h

Plasmidisolation of pSB1C3_barnaseB c5, 22, 30, 31, 32, 45

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

Plasmid DNA purification kit (Zymo PURE)

Nucleo spin plasmid protocol

PCR of KanR & pSB1C3

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

PCR

Q5 High-fidelily (NEB) protocol
Primer: 17nx & 17ny (Kan)
Primer: 17od & 17oe (pSB1C3)
Denaturation: 54 °C & 60 °C
Elongation: 30 s & 75 s

Restriction Digest

5 µL 10x CutSmart buffer
1 µg plasmid
1 µL DpnI
filled up to final volume 50 µL with ddH20
Incubation: ON, 37 °C
Inactivation: 20 min, 80 °C

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of the positive clone 3 of BBa_K2201200

Procedure:

standard procedure

1,25µl Cam in 5mL LB

Restriction digest of KanR fragment

Investigators: Olga Schmidt

Superior experiment: Construction of selection plasmid

Procedure:

Restriction Digest

5 µL NEB2.1 buffer
2 µL DNA
1 µL XbaI
1 µL PstI
filled up to final volume 50 µL with ddH20
Incubation: 60 min, 37 °C
Heatinactivation: 20 min, 80 °C

Aim of the Experiment

Investigators: Yannic Kerkhoff

Superior experiment: GoTaq PCR of 32 clones of BBa_K2201200

Procedure:

Screening for positive transformations containing the Biobrick

standard GoTaq-protocol

template: colonie pcr clones of GA_K2201200
primer fwd: 17ns
primer rev: 17nt
annealing temperature: 56°C
extension time: 40 seconds

positive clone 20

Native PAGE of annealed primers (17jl & 17jm)

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

Native DNA PAGE standard protocol

tested samples:

HEPES annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM
Aqua annealing (17jl & 17jm): 100 µM, 10 µM, 5 µM, 2.5 µM, 1 µM, 0.5 µM

Variation: 60 V, 2 h

multiplication of plasmids containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: preculture of BL21(DE3) BBa_K2201321

Procedure:

standard procedure

1,25µl Cam in 5mL LB

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K2201321 clone 3

Procedure:

standard protocol

319,1 ng/µL

Native Page of annealed primers (17jl & 17jm; 17jh & 17ji)

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

Native DNA PAGE standard protocol

tested samples:

HEPES annealing (17jl & 17jm): 0.5 µM
Aqua annealing (17jl & 17jm): 0.5 µM
Composite Buffer annealing (17jh & 17 jm): 0.5 µM
all Primers (ssDNA): 1 µM

2017-08-28 - 2017-09-03

Native PAGE of annealed primers (HEPES annealing & Composite Buffer annealing) of the same concentration to test if all annealings have the same quality

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system - Primer annealing test

Procedure:

all samples were diluted to a final concentration of 0.5 µM
Native DNA PAGE standard protocol

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of the Biobrick Assembly product CP3

Procedure:

standard heatshock protocol

used own produced chemo competend BL21(DE3)
streaked out on LB-plates with Cam

Restriction digest of control annealings (mutC)

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Restriction Digest (MnlI) of aqua annealing sample: mutC (0.5 µM)

2 µL MnlI
5 µL CutSmart
25 µL mutC dsDNA
18 µL nuclease-free H2O
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Native DNA PAGE standard protocol

Tested samples:

digested mutC sample
native mutC sample
ssDNA primers (17jl & 17jm)

GoTaq PCR of 14 clones of BBa_K2201322

Investigators: Yannic Kerkhoff

Superior experiment: Screening for positive transformations containing the Biobrick

Procedure:

standard GoTaq-protocol

template: colonie pcr clones of BBA_K2201322
primer fwd: Präfix_fwd
primer rev: Suffix_rev
annealing temperature: 56°C
extension time: 40 seconds

positive: clone 3, 4, 5

Restriction digest of aqua & HEPES annealing

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Restriction Digest (MnlI) of aqua & HEPES annealing sample: mutC (0.5 µM)

2 µL MnlI
5 µL CutSmart
25 µL mutC dsDNA
18 µL nuclease-free H2O
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Native DNA PAGE standard protocol

Tested samples:

digested mutC sample (aqua & HEPES annealing)
native mutC sample
ssDNA primers (17jl & 17jm)

Aqua annealing of mutA, mutT, mutG and mutC

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Aqua annealing standard protocol (final concentration: 0.5 µM)

mutA: 17jf & 17jg
mutT: 17jj & 17jk
mutG: 17jh & 17ji
mutC: 17jl & 17jm

final volume of each sample: 50 µL

2017-09-04 - 2017-09-10

Restriction digest of control annealings (mutA, mutT, mutG, mutC) and aqua annealing of oligo2

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Oligo2 annealing

Aqua annealing standard protocol (final concentration: 0.5 µM)

Restricted samples:

MnlI -> mutC, oligo2
SapI -> mutG, oligo2
BsaI -> mutT, oligo2
EciI -> mutA, oligo2

Restriction Digest:

1 µL Enzyme
5 µL CutSmart
25 µL mutC dsDNA
19 µL nuclease-free H2O
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Native DNA PAGE standard protocol

SDS-Page and western blot of BBa_K2201321

Investigators: Yannic Kerkhoff

Superior experiment: SDS-Page and western blot of BBa_K2201321

Procedure:

standard SDS protocoll
standard Western blot protocoll

integration of the mRFP (BBa_J04450) in the aaRS plasmid as optical controle for the integration of an randomerized dsDNA

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

amplification of the mRFP (Phusion Master Mix), Primer vg, vh, annealing temperature: 60°C
amplification of the library backbone(Phusion Master Mix), Primer 17ve, 17vf, Annealing temperature: 61°C
Gibson Assembly with Gibson Master Mix + mRFP (3.1 µL) + library backbone (1.9 µL)
transformation in DH5a via heatshock

Generating the randomerized dsDNA for the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

ssDNA Annealing protocoll with the primers 17vi (1 µL), 17vj (1,4 µL)
agarose gelelectrophoresis, 3 %, positive result: bands of 120 bp

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the optical controle
transformation in DH5a via heatshock

2017-09-11 - 2017-09-17

Annealing, restriction digest and native PAGE of all samples

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Aqua annealing standard protocol (final concentration: 0.5 µM)

mutA: 17jf & 17jg
mutT: 17jj & 17jk
mutG: 17jh & 17ji
mutC: 17jl & 17jm
oligo2: olgio2_sense & oligo2_antisense

final volume of each sample: 50 µL
Restricted samples:

MnlI -> mutC, oligo2
SapI -> mutG, oligo2
BsaI -> mutT, oligo2
EciI -> mutA, oligo2

Restriction Digest:

1 µL Enzyme
5 µL CutSmart
25 µL mutC dsDNA
19 µL nuclease-free H2O
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Native DNA PAGE standard protocol

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the optical controle (8 x)
transformation in DH5a via heatshock (8 x)

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (16 x)
transformation in DH5a via heatshock (16 x)

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
transformation in DH5a via heatshock (25 x)

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
transformation in DH5a via heatshock (25 x)

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
transformation in DH5a via heatshock (25 x)

Annealing, restriction digest and native PAGE of all samples

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Aqua annealing standard protocol (final concentration: 0.5 µM)

mutA: 17jf & 17jg
mutT: 17jj & 17jk
mutG: 17jh & 17ji
mutC: 17jl & 17jm
oligo2: olgio2_sense & oligo2_antisense

final volume of each sample: 2x50 µL
Restricted samples:

MnlI -> mutC
SapI -> mutG, oligo2
BsaI -> mutT
EciI -> mutA

Restriction Digest:

1 µL Enzyme
5 µL CutSmart
25 µL mutC dsDNA
19 µL nuclease-free H2O
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Native DNA PAGE standard protocol

2017-09-18 - 2017-09-24

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of the EutS-part from CU Boulder

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with Amp

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock transformation of the FusionRedTag-part from CU Boulder

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with Kan

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock cotransformation of the EutS-part and FusionRedTag-part from CU Boulder

Procedure:

standard heatshock protocol

used own produced chemo competend DH5alpha
streaked out on LB-plates with AmpKan

Agarose gel electrophoresis of digested mutA, mutT, mutG & mutC

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Agarose gel electrophoresis (2%)

60 V, 40 min

Annealing, restriction digest and native PAGE of all samples

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Aqua annealing standard protocol (final concentration: 0.5 µM)

mutA: 17jf & 17jg
mutT: 17jj & 17jk
mutG: 17jh & 17ji
mutC: 17jl & 17jm
oligo2: olgio2_sense & oligo2_antisense

final volume of each sample: 2x50 µL
Restricted samples:

MnlI -> mutC
SapI -> mutG, oligo2
BsaI -> mutT
EciI -> mutA

Restriction Digest:

1 µL Enzyme
5 µL CutSmart
25 µL mutC dsDNA
19 µL nuclease-free H2O
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Native DNA PAGE standard protocol

Combination of two biobricks to get the part BBa_K2201200 in a tetracycline low copy plasmid

Investigators: Yannic Kerkhoff

Superior experiment: Biobrick Assembly of BBa_K2201200 in psB3T5

Procedure:

Standard BioBrick Assembly protocol

Backbone: pSB3T5, 2,0µL
upstream: BBa_K2201200, 3,6µL
Ligation time was 12 h at 8°C

cultivation

glycerine stocks of K2201200, K2201220, K2201221, K2201320, K2201321

Investigators: Yannic Kerkhoff

Superior experiment: glycerine stocks of K2201200, K2201220, K2201221, K2201320, K2201321

Procedure:

glycerine stock standard protocol

Screening for positive transformations containing the Biobrick

Investigators: Yannic Kerkhoff

Superior experiment: GoTaq PCR of 30 clones of K2201200 in pSB3T5

Procedure:

standard GoTaq-protocol

template: colonie pcr clones of K2201200 in pSB3T5
primer fwd: Präfix_fwd
primer rev: Suffix_rev
annealing temperature: 56°C
extension time: 40 seconds

positive clones 1, 3, 6

Preculture

Colony PCR of pSB3K5_mRFP_link_ccdB and pSB1C3_PtNTT2(31-575)-GFP

Investigators: Camilla Maerz

Superior experiment: Construction of pSB1C3-PlacUV5_PtNTT2

Procedure:

PCR

Go Tag (Promega) protocol
Primer: VR, VF2
Danaturation: 56 °C
Elongation: 90 s & 180 s

Agarose gel electrophoresis (1%)

100 V, 30 min

2017-09-25 - 2017-10-01

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock cotransformation of K2201200 in pSB3T5 and K2201321

Procedure:

standard heatshock protocol

used own produced chemo competend BL21(DE3)
streaked out on LB-plates with TetCam

getting plasmids containing the Biobrick for further experiments

Investigators: Yannic Kerkhoff

Superior experiment: plasmid isolation of preculture of BBa_K2201207

Procedure:

standard protocol

75,6 ng/µL

Biobrick Assembly

Aqua annealing of mutA-1, mutG-1, mutC-1, mutG-1 & oligo1

Investigators: Camilla Maerz

Superior experiment: M.A.X restriction system

Procedure:

Aqua annealing standard protocol (final concentration: 0.5 µM)

mutA-1: 17iv & 17iw
mutT-1: 17jb & 17jc
mutG-1: 17ix & 17iy
mutC-1: 17jd & 17jc
oligo1: olgio1_sense & oligo1_antisense

final volume: 50 µL

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock cotransformation of K2201240 and K2201207

Procedure:

standard heatshock protocol

used own produced chemo competend BL21(DE3)
streaked out on LB-plates with AmpCam

getting positive clones for further work

Investigators: Yannic Kerkhoff

Superior experiment: heatshock cotransformation of K2201241 and K2201207

Procedure:

standard heatshock protocol

used own produced chemo competend BL21(DE3)
streaked out on LB-plates with AmpCam

Q5-PRC + Gel + cleanUp

2017-10-02 - 2017-10-08

UBP-PCR with TiTaq

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutA, mutT, mutG, mutC and oligo 2 (1 ng/µL)

Primer: 17vt & 17vu
Primer annealing: 56 °C
Elongation: 20 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

Restriction Digest

1 µL Enzyme
5 µL CutSmart
25 µL mutC dsDNA
19 µL nuclease-free H2O
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Restricted samples:

MnlI -> mutC
SapI -> mutG
BsaI -> mutT
EciI -> mutA
EciI, BsaI, SapI, MnlI -> oligo2
Variation: oligo2 was digested with 0.5 µL of each enzyme

Agarose gel electrophoresis (2 %)

100 V, 20 min

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
transformation in DH5a via heatshock (25 x)

Kanymycin with amber stop codon

Investigators: Laura Schlueter

Superior experiment: positiv selection plasmid

Procedure:

amplification (~850 bp) of the kanamycin with primers 17jo, 17jp (no second MET codon) 71°C
amplification (~850 bp) of the kanamycin with primers 17jr, 17jp (two amber stop codon) 71°C

amplification of the K608007 backbone with the primers 17hb, 17hd 57°C

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
transformation in DH5a via heatshock (25 x)

UBP-PCR with GoTaq G2 and TiTaq

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR with TiTaq:

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutA, mutT, mutG, mutC and oligo 2 (5 ng/µL)

Primer: 17 vt & 17 vu
Primer annealing: 56 °C
Elongation: 20 s

PCR with TiTaq:

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutA, mutT, mutG and mutC (5 ng/µL)

Primer: VR & VF2
Primer annealing: 56 °C
Elongation: 20 s

PCR with GoTaq:

PCR-UBP standard protocol (GoTaq)
samples:

Ligation of: mutA, mutT, mutG, mutC and oligo2 (5 ng/µL)

Primer: 17 vt & 17 vu
Primer annealing: 56 °C
Elongation: 20 s

cloning of the aaRS in pSB3C5 (low copy)

Investigators: Laura Schlueter

Superior experiment: aaRS growth experiment

Procedure:

restriction of pSB3C5 and aaRS+tRNA (NPA, AcF, Prk) with EcoRI and PstI following the restriction protocol
extraction from the gel (Macherey-Nagel Kit)
ligation of pSB3C5 and aaRS+tRNA (NPA, AcF, Prk) with T4 Ligase (NEB)
Trafo via heatshock

sceening of the kanamycin (03.10.2017)

Investigators: Laura Schlueter

Superior experiment: positiv selection plasmid

Procedure:

Colony PCR (GoTaq, Primer VF2, VR, 56.5 °C)

Restriction digest of ligated oligo2

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

Restriction Digest

5 µL CutSmart
30 µL ligated oligo2 DNA
2 µL EcoRV
filled up to final volume 50 µL with ddH20
Incubation: 120 min, 37 °C
Inactivation: 20 min, 65 °C

Gradient PCR of mutT

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR
PCR-UBP standard protocol (GoTaq G2)

sample: mutT (5 ng/µL)
Primer: 17 vt & 17 vu
Primer annealing: 50 - 56 °C
Elongation: 20 s

Agarose gel electrophoresis (2%)

100 V, 30 min

PCR of mutA, mutT, mutG & mutC with UBP and oligo2 without UBP

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR with TiTaq:

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)

Primer: VR & VF2
Primer annealing: 56 °C
Elongation: 20 s
Variation:

oligo2 without UBP, instead 1 µL H20 was added
0.5 µL isoG & 0.5 µL isoCMe were added to mutA, mutT, mutG and mutC

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS Plasmid

Procedure:

plasmid isolation of the Tyr-RS library-mix

we stamped out the not randomized, negative, clones, easy to identify by the red colour because of the mRFP
washed from the cultivation plates with LB-media

restriction of the Library with EcoRI and PstI
proof of the restriction via agarose-gelelectrophoresis, 1 %

positve result: bands on ~2000 bp (backbone) and ~1000 bp (Tyr-RS with NNK)

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
transformation in DH5a via heatshock (25 x)

2017-10-09 - 2017-10-15

PCR with switched primers: VF2 & 17vu, VR & 17vt

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR with TiTaq:

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)

Primer: VF2 & 17vu, VR & 17vt
Primer annealing: 56 °C
Elongation: 20 s

PCR with GoTaq:

PCR-UBP standard protocol (GoTaq)
samples:

Ligation of: mutA, mutT, mutG and mutC (5 ng/µL), oligo2 (25 ng/µL)

Primer: VF2 & 17vu, VR & 17vt
Primer annealing: 56 °C
Elongation: 20 s

Agarose gel electrophoresis (2%)

100 V, 30 min

Generating the library

Investigators: Laura Schlueter

Superior experiment: aaRS plasmid

Procedure:

Gibson Assembly Master Mix + dsDNA (3.5 µl) + library Plasmid (0.5 µL) with the opotical controle (25x)
transformation in DH5a via heatshock (25 x)

PCR with higher DNA template concentrations and gradient PCR of mutT

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR with TiTaq:

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutT (5 ng/µL) and oligo2 (50 ng/µL)

Primer: VF2 & 17vu
Primer annealing: 56 °C
Elongation: 20 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

Restriction Digest

0.5 µL Enzyme
2.3 µL CutSmart
20 µL dsDNA
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Restricted samples:

BsaI -> mutT
EciI, BsaI, SapI, MnlI -> oligo2
Variation: oligo2 was digested with 0.5 µL of each enzyme

Agarose gel electrophoresis (2 %)

100 V, 20 min

Gradient PCR with TiTaq:

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutT (5 ng/µL)

Primer: VF2 & 17vu
Primer annealing: 50 - 58 °C
Elongation: 20 s

Agarose gel electrophoresis (2%)

100 V, 30 min

PCR of mutA, mutT, mutG, mutC and oligo2

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR with GoTaq:

PCR-UBP standard protocol (GoTaq)
samples:

Ligation of: mutA, mutT, mutG, mutC (5 ng/µL) and oligo2 (25 ng/µL)

Primer: VF2 & 17vu
Primer annealing: 56 °C
Elongation: 20 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

Restriction Digest

1 µL Enzyme
2.3 µL CutSmart
20 µL dsDNA
Incubation: 37 °C, 12 h
Inactivation: 20 min., 65 °C

Restricted samples:

MnlI -> mutC
SapI -> mutG
BsaI -> mutT
EciI -> mutA
EciI, BsaI, SapI, MnlI -> oligo2

Agarose gel electrophoresis (2 %)

100 V, 30 min

positive selection with the library

Investigators: Laura Schlueter

Superior experiment: pos selection

Procedure:

using cultivation plates with kan (0.5 mM), Cm (0.25 mM), Tet (0.5 mM)

no growth

Variation of restriction digest

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

Restriction Digest

3 µL Enzyme
2.5 µL CutSmart
20 µL dsDNA
Incubation: 37 °C, 1 h
Inactivation: 20 min., 65 °C

Restricted samples:

MnlI -> mutC
SapI -> mutG
BsaI -> mutT
EciI -> mutA
EciI, BsaI, SapI, MnlI -> oligo2

Agarose gel electrophoresis (2 %)

100 V, 30 min

positive selection with the library

Investigators: Laura Schlueter

Superior experiment: pos selection

Procedure:

Transformation (heatshock) of the library plasmid mix in competent cells containing the positive selction plasmid
using cultivation plates with kan (0.3 mM), Cm (0.25 mM), Tet (0.5 mM)

no growth

positive selection with the library

Investigators: Laura Schlueter

Superior experiment: pos selection

Procedure:

Transformation (heatshock) of the library plasmid mix in competent cells containing the positive selction plasmid
extended regeneration time: adding 0,5 mM IPTG after 1 h in SOC media
using cultivation plates with kan (0.3 mM), Cm (0.25 mM), Tet (0.5 mM), IPTG (0,5 mM)

no growth

growth experiments with the aaRS

Investigators: Laura Schlueter

Superior experiment: pos selection

Procedure:

comparing AcF-TAG, AcF-Leu, Cou-AS, Prk, NPA (on low and high copy plasmid) pSB1C3, pSB3T5

cultivation:

LB media, Cm (0.25 mM) / Tet (0.5 mM) and 1 mM of the ncAA
12 microtiter well plate, 600 rpm, 37°C

2017-10-16 - 2017-10-22

PCR with TiTaq and Q5-polymerase

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

PCR with TiTaq:

PCR-UBP standard protocol (TiTaq)
samples:

Ligation of: mutA, mutT, mutG & mutC (5 ng/µL) and oligo2 (50 ng/µL)

Primer: VF2 & 17vu
Primer annealing: 56 °C
Elongation: 20 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

PCR with Q5 High-Fidelity Polymerase-polymerase (per reaction, 25 µL):

5 µL Reaction buffer
1.25 µL dNTPs (2 mM)
1.25 µL 3'-Primer (10 mM)
1.25 µL 5'-Primer (10 mM)
1 µL DNA template
0.25 µL Q5 Hifi-Polymerase
14 µL H20
mutA, mutT, mutG, mutC: + 1 µL H20
oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
Primer: VF2 & 17vu
Primer annealing: 56 °C
Elongation: 10 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

Restriction Digest

1 µL Enzyme
20 µL dsDNA
2.3 µL CutSmart Buffer
Incubation: 37 °C, 15 h (BsaI & MnlI), 2 h (SapI & EciI)
Inactivation: 65 °C, 20 min

Restricted samples:

MnlI -> mutC
SapI -> mutG
BsaI -> mutT
EciI -> mutA
EciI, BsaI, SapI, MnlI -> oligo2

PCR with different polymerases

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

samples:

Ligation of: mutA, mutT, mutG & mutC (5 ng/µL) and oligo2 (50 ng/µL)

PCR with Allin Hifi DNA Polymerase (per reaction, 25 µL):

5 µL ReactionBuffer
0.25 µL Hifi DNA Polymerase
2.5 µL 3'-Primer (10 mM)
2.5 µL 5'-Primer (10 mM)
1 µL DNA template
12.75 µL H20
mutA, mutT, mutG, mutC: + 1 µL H20
oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
Primer: VF2 & 17vu
Primer annealing: 56 °C
Elongation: 10 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

PCR with innuDRY Standard PCR MasterMix (per reaction, 20 µL):

10 µL innduDRY Mastermix
2 µL 3'-Primer (10 mM)
2 µL 5'-Primer (10 mM)
1 µL DNA template
5 µL H20
mutA, mutT, mutG, mutC: + 1 µL H20
oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
Primer: VF2 & 17vu
Primer annealing: 54 °C
Elongation: 30 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

PCR with BioMaster-HS Taq PCR Color (per reaction, 25 µL):

12.5 µL Mastermix
0.7 µL 3'-Primer (10 mM)
0.7 µL 5'-Primer (10 mM)
1 µL DNA template
10.1 µL H20
mutA, mutT, mutG, mutC: + 1 µL H20
oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
Primer: VF2 & 17vu
Primer annealing: 51 °C
Elongation: 20 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

PCR with Fire Polymerase (per reaction, 20 µL):

4 µL Mastermix
0.5 µL 3'-Primer (10 mM)
0.5 µL 5'-Primer (10 mM)
1 µL DNA template
14 µL H20
mutA, mutT, mutG, mutC: + 1 µL H20
oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
Primer: VF2 & 17vu
Primer annealing: 56 °C
Elongation: 20 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

PCR with Phusion polymerase (per reaction, 25 µL):

12.5 µL Mastermix
1.25 µL 3'-Primer (10 mM)
1.25 µL 5'-Primer (10 mM)
1 µL DNA template
9 µL H20
mutA, mutT, mutG, mutC: + 1 µL H20
oligo2: + 0.5 µL isoCme, + 0.5 µL isoG
Primer: VF2 & 17vu
Primer annealing: 56 °C
Elongation: 20 s

PCR and DNA purification kit (Macherey-Nagel)

Nucleo spin PCR and Monarch® PCR & DNA Clean-up protocol

Restriction Digest

1 µL Enzyme
20 µL dsDNA
2.3 µL CutSmart Buffer
Incubation: 37 °C, 15 h (BsaI & MnlI), 2 h (SapI & EciI)
Inactivation: 65 °C, 20 min

Restricted samples:

MnlI -> mutC
SapI -> mutG
BsaI -> mutT
EciI -> mutA
EciI, BsaI, SapI, MnlI -> oligo2

PCR with different polymerases

Investigators: Camilla Maerz

Superior experiment: PCR with UBP

Procedure:

Agarose gel electrophoresis with all samples (17.10)
Agarose gel electrophoresis (2%)

100 V, 40 min