Transformation of E. coli XL1 blue with EreA and EreB (pDEST14)

Investigator: Jeff, Leonie, Florian

Aim of the experiment: Transformation of E. coli XL1 blue with EreA and EreB (pDEST14).

Procedure:

• Plasmid DNA was received dried in paper from McMaster University.

• DNA was resuspended in ddH2O

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Investigator: Leonie

Aim of the experiment: Miniprep of pRK792, pRK793, pRIL, ereA, ereB

Procedure:

• Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

• Concentrations were determined by measuring the absorption:

• The procedure will have to be repeated for pRIL

Transformation of E. coli XL1 blue with PSH21 (P17)

Investigator: Johanna, Louise

Aim of the experiment: Transformation of E. coli XL1 blue with PSH21.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.

• 5 µl of DNA was added to 250 µl of competent cells and gently mixed.

• 60 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding the DNA to 2 ml LB-medium.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry

Investigator: Andreas

Aim of the experiment: Transformation of E. coli XL1 blue with pSB1C3-GFP, pSB1C3-mkate2, pSB1C3-mCherry.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice for 10 minutes.

• 5 µl of DNA was added to 250 µl of competent cells and gently mixed.

• 60 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding the DNA to 2 ml LB-medium.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• The transformated cells were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated on chlorampenicol and ampicillin plates.

PCR, purification and analytical geleletrophoresis of EreA (P15) and EreB (P16)

Investigator: Jeff, Louise, Florian, Rosario, Andi, Johanna

Aim of the experiment: PCR of EreA (P15) and EreB (P16).

Procedure:

Operational sequence:

• PCR reaction mixture

• Mix with pipette

• The gradient PCR program was performed after following scheme with following conditions (T_m=54 °C; ΔG=2 °C; P15 in row 11(=52 °C); P16 in row 1 (=54.0 °C)):

• After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

• 9 µl of the purified PCR products were mixed with 1 µl DNA loading buffer (10x) for analytical gelelectrophoresis

• Analytical gelelectrophoresis was performed at 90 V for 60 min in 1% agarose gel.

Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI

Investigator: Jeff, Andi, Florian, Rosario, Louise

Aim of the experiment: Preparative digestion and gelelectrophoresis of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.

Procedure:

• Batch for preparative digestion of PCR product of EreA (F1) and EreB (F2) with XbaI & AgeI.

• Batch for preparative digestion of P52 for preperation of pSB1C3 vector:

• Incubation for 3 h at 37 °C.

• 5 µl of DNA loading buffer (10x) were added to the reaction batches for F1 & F2 and 4 µl of DNA loading buffer (10x) were added to the reaction batches for Pxx after digestion and were loaded on an 1% agarose gel for preparative gelelectrophoresis.

• Preparative gelelectrophoresis was performed at 90 V for 60 min.

• Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3)

Investigator: Jeff, Andi, Florian, Rosario, Louise

Aim of the experiment: Ligation of F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3). Quickchanges have still to be performed.

Procedure:

Ligation batch for F8+F6 (EreA in pSB1C3), F8+F7 (EreB in pSB1C3):

• Ligation batch for F8+F6

• Ligation batch for F8+F7

• Negative control was als prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batches.

• Cycled ligation has been performed after following protocol in a thermocycler:

Lid temperature = 37 °C

Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB)

Investigator: Florian, Jeff, Leonie, Louise, Katrin, Ingmar

Aim of the experiment: Transformation of E. coli XL1 blue with F8(pSB1C3)+F6(EreA) and F8(pSB1C3)+F7(EreB). Quickchanges has now to be performed to remove forbidden restriction sites.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 10 µl of ligation products F8+F6, F8+F7, F8 negative control were added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs

Investigator: Andi, Jeff, Rosario

Aim of the experiment: Deletion of found mutations.

Operational sequence

1. PCR general setup

Reaction batch 1

Reaction batch 2

PCR cycling parameters

• Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied.

• After the PCR was finished 1 µl of the DpnI restriction enzyme (10 U/µl)was added

• Now the reaction was mixed gently and thoroughly, spinned down in a microcentrifuge for 1 minute, and immediately incubated at 37 °C for 1 hour to digest the parental supercoiled dsDNA

2. Used constructs and primers

Picking of transformed Plasmids F8 + F6

Investigator: Andi

Aim of the study: Picking from the overnight transformation of the ligated F8+F6, to see whether ligation is successful or not.

Operational sequence:

• Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA)

Investigator: Louise

Aim of the experiment: Miniprep of overnight culture of transformated E.~coli XL1-Blue with F8 (pSB1C3) + F6 (EreA) of 2 clones picked the day before

Procedure:

• Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)
• The plasmids were labelled as P63 and P64

Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI

Investigator: Louise

Aim of the experiment: Analytical digestion and gelelectrophoresis of P63 and P64 with AgeI-HF and XbaI

Procedure:

• Batch for analytical digestion of P63 and P64 with AgeI-HF and XbaI

• Incubation for 90 min at 37 °C.

• Analytical gelelectrophoresis was performed at 90 V for 60 min.

Preparative digestion of PCR product of EreB (F2) with XbaI & AgeI

Investigator: Louise,Leonie,Johanna

Aim of the experiment: Preparative digestion of PCR product EreB (F2) with XbaI & AgeI (second try since there did not grow any transformed bacteria in the first time).

Procedure:

• Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.

• Incubation for 3 h at 37 °C
• The digested fragment was labelled F14

Purification and ligation of digested PCR product EreB (F14)

Investigator: Louise, Rosario

Aim of the experiment: PCR Purification and ligation of digested PCR product EreB (F14).

Procedure: PCR Purification of EreB PCR (F14)

• To purify the digested Fragment(F14) the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be 5 ng/µl via Nano drop.

Ligation of F14 + F8(pSB1C3)

• Ligation batch for F8+F14 (positive control)

• Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
• Cycled ligation has been performed after following protocol in a thermocycler:

Lid temperature = 37 °C

Transformation of ligation products of F10 + F11 into E.coli Xl1-Blue

Investigator: Andi

Aim of the experiment:Transformation of the ligation products of F10 + F11 and the negative control in pTUM104 in XL1 Blue.

Operation Sequence

• melting of 2x 200 µl Ca-competent E.coli XL1-Blue cells on ice
• Preparing 4 Tubes with 100 µl of Ca-competent E.coli XL1-Blue cells
• addition of 5 µl of the ligation products
• incubation for 30 min on ice
• heat shock for 5 min at 37 °C
• transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
• plate 100 µl on an Amp-LB-plate
• sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Transformation of Quickchange Product P62 into E.coli Xl1-Blue

Investigator: Andi

Aim of the experiment:Transformation of the Quickchange Product P62 and the negative control in XL1 Blue.

Operation Sequence

• melting of 1x 200 µl Ca-competent E.coli XL1-Blue cells on ice
• Preparing 4 Tubes with 50 µl of Ca-competent E.coli XL1-Blue cells
• addition of 1 µl of the Quickchange Product
• incubation for 30 min on ice
• heat shock for 5 min at 37 °C
• transfer of cells to 1 ml LB-medium without antibiotics and incubate at 37°C and 180 rpm for 30 min
• plate 100 µl on an Amp-LB-plate
• sediment the leftover in a centrifuge (30 - 60 sec, 13 000 rpm) and resuspend the sediment in 100 µl LB-medium and plate it as well on an Amp-LB-plate

Transformation of E. coli XL1 blue with ligation products F8+F14 (EreB in pSB1C3, RFC25)

Investigator: Jeff, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with ligation product F8+F14 (EreB in pSB1C3, RFC25). Further quickchanges still have to be done.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 10 µl of the ligation products were added to 200 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Picking of transformed Plasmid F10+F11 (IRES from polio virus, blunt ligated with pSB1C3 for sequencing)

Investigator: Andi

Aim of the study: Picking of transformed Plasmid F10+F11 (AlcR blunt ligated with pSB1C3 for sequencing).

Operational sequence:

• Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

• 10 colonies were picked.

Analyzing sequencing results

Investigator: Leonie,Johanna

Aim of the experiment: Comparison of the sequencing results with the Biobrick (BB) sequences.

Procedure: Using Geneious to align the sequencing results with the BB sequences.

LMU GFP in RFC 25(p20)

• The provided sequence in the parts registry is totally wrong.
• The comparison of the amino acid seqeunces reveals several point mutations.

FluA in RFC 25 (p45)

• Did not yield sequencing results (does the primer bind ?)
• No presence of RCF 25 standard restriction enzymes

mCherry in RFC 25 (p46)

• Did not yield sequencing results (does the primer bind ?)
• No presence of RCF 25 standard restriction enzymes

CaMV35S in RFC 25 (p23)

• high identity
• high match with HQ699544 (Blast)

LMU mKate2in RFC 25 (p21)

• no continous ORF
• Did not yield sequencing results

DREB1C promoter in RFC 25 (p26)

• Present of fluorecent protein gene

xylE in RCF 25 (p38)

• Did not yield sequencing results

SV40 in RCF 25 (p28)

• sequencing result agrees with the laccases
• the sequencing result can't be the one of SV40

Quickchange mutagenesis SDMI of p27

Investigator: Rosario, Jeff, Flo, Andi

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 65.

Procedure: PCR

Reaction batch 1

Reaction batch 2

PCR cycling parameters

• Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles was increased to 20
• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Quickchange mutagenesis SDMI of p38

Investigator: Jeff, Flo, Andi, Rosario

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled 67.

Procedure: PCR

Reaction batch 1

Reaction batch 2

PCR cycling parameters

• Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, but the amount of cycles have been increased to 20
• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Quickchange mutagenesis SDMI of P63

Investigator: Jeff, Rosario, Flo, Andi

Aim of the experiment: Sequencing results showed a forbidden restriction site in this vector. The sdm will delete this restricition site. The resulting Plasmid was labeled P66.

Procedure: PCR

Reaction batch 1

Reaction batch 2

PCR cycling parameters

• Having completed the PCR cycling parameters listed above both PCR reaction batches were mixed together and the cycling parameters listed above were one time more applied, the amount of cycles was raised to 20
• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the PCR batch and incubate for 1 h at 37 °C.

Transformation of E. coli XL1 blue with P65

Investigator: Jeff, Rosario, Johanna, Andi, Flo, Leonie

Aim of the experiment: Transformation of E. coli XL1 blue with P65 .

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P66

Investigator: Jeff, Rosario, Flo, Johanna, Andi, Leonie

Aim of the experiment: Transformation of E. coli XL1 blue with P66.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Transformation of E. coli XL1 blue with P67

Investigator: Jeff, Rosario, Johanna, Andi, Leonie, Flo

Aim of the experiment: Transformation of E. coli XL1 blue with P67.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Sequencing of P63 & P64

Investigators: Louise

Aim of the experiment: Sequencing of genes for which no prior sequencing results were available.

Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI

Investigator: Louise,Leonie,Johanna

Aim of the experiment:Preparative digestion of psB1C3-RFP-generator (P60) and another psB1C3 (P8) as well as P64 (EreA in defect psB1C3 (F8)) with XbaI & AgeI to recover EreA and prepare two new vectors for cloning.

Procedure:

• Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.

• Incubation for 2.5 h at 37 °C
• The digested fragments were labelled F17(P8), F18(P60), F19(P64)

PCR, purification and analytical geleletrophoresis of and EreB (P16)

Investigator: Jeff, Louise

Aim of the experiment: PCR of EreB (P16).

Procedure:

Operational sequence:

• PCR reaction mixture

• Mix with pipette

• The PCR program TMKS was performed after following scheme:

• After PCR, the product was purified with QIAquick PCR Purification Kit, Qiagen.

• 4 µl of the purified PCR products were mixed with 0.4 µl DNA loading buffer (10x) for analytical gelelectrophoresis

• Analytical gelelectrophoresis was performed at 90 V for 30 min in 1% agarose gel.

Purification and ligation of digested products F17, F18, F19

Investigator: Jeff, Louise

Aim of the experiment: Purification and ligation of digested products F17, F18, F19.

Procedure:

• To purify the digested Fragments the QIAquick PCR Purification Kit was used. The concentration of the purified product was found to be XX ng/µl via Nano drop.

Ligation of F19 + FXX(pSB1C3)

• Ligation batch for F19+FXX (positive control)

• Negative control was also prepared. Instead of the insert, the same volume of ddH2O was added to the reaction batch.
• Cycled ligation has been performed after following protocol in a thermocycler:

Preparative digestion of ereB (P64) with XbaI & AgeI.

Investigator: Jeff, Louise, Katrin

Aim of the experiment:Preparative digestion of EreB (P64) with XbaI & AgeI.

Procedure:

• Batch for preparative digestion of PCR product and EreB (F2) with XbaI & AgeI.

• Incubation for 2.5 h at 37 °C
• The digested fragments were labelled

Quick Change mutagenesis to remove mutations found by sequencing in our gene constructs

Investigator: Jeff, Leonie

Aim of the experiment: removal of forbidden restiction sites from laccase

Operational sequence

PCR general setup

PCR cycling parameters

Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

Investigator: Jeff, Andi, Johanna, Leonie

Aim of the experiment: Transformation of E. coli XL1 blue with the 4 Quickchangeproducts using 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 5 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on chloramphenicol plates 1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was discarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on chloramphenicol plates (1: O26 + O27, 2: O28 + O29, 3: O30 + O31, 4: O32 + O33)

Analytical digestion and analytical gelelectrophoresis of F17+F19, F17+F20, F17+F21 and F22+F23

Investigator: Jeff, Andi

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of ligation products F17+F19, F17+F20, F17+F21, F22+F23.

Procedure:

• Mastermix for analytical digestion of F17+F19, F17+F20, F17+F21 with XbaI+PstI-HF:

• Batch for digestion of F22+F23 with EcoRI-HF:

• Incubation for 90 min at 37 °C.

• Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.

Results:

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi

Aim of the experiment: Transformation of E. coli XL1 blue with P28, P102 and P100.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange (SDMI) Product of P28

Investigator: Jeff, Andi, Rosario, Johanna, Leonie

Aim of the experiment: Miniprep of overnight culture of transformated E. coli XL1 blue with the first Quickchange Product of P28. 8 clones were picked the day before.

Procedure:

• Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and analytical gelelectrophoresis of P28, P117, P118, P119, P120, P121, P122, P123

Investigator: Rosario, Andi, Leonie, Johanna, Jeff

Aim of the experiment: Analytical digestion and analytical gelelectrophoresis of P28 (promoter SV40) and quickchange products P117, P118, P119, P120, P121, P122, P123 (all QC - SDMI - of Bpul Laccase to remove forbidden AgeI).

Procedure:

• Mastermix for analytical digestion of P28, P117, P118, P119, P120, P121, P122, P123 AgeI-HF:

• Incubation for 90 min at 37 °C.

• Analytical gelelectrophoresis was performed two times for each ligation product at 90 V for 60 min.

Results:

Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P28 (Laccase), P106 (EreB)

Investigator: Jeff, Andi, Johanna, Rosario, Leonie

Aim of the experiment: Removal of forbidden restiction sites from P28 (Laccase), P106 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P106

PCR cycling parameters - P106

• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Reaction batch - P116 (Laccase)

PCR cycling parameters - P116

• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange products with P28, P100 and P102 into E. coli XL1 blue

Investigator: Jeff, Andi, Johanna, Rosario

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P106 and P116.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Quick Change mutagenesis to remove forbidden restriction sites - SDMII - of P116 (Laccase), P109 (EreB)

Investigator: Volker, Louise, Katrin, Leonie, Flo

Aim of the experiment: Removal of forbidden restiction sites from P116 (Laccase), P109 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P109

PCR cycling parameters - P109

• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Reaction batch - P116 (Laccase)

PCR cycling parameters - P116

• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange products into E. coli XL1 blue

Investigator: Volker

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM II of P109 and P116.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 2 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium to each tube.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.

Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful

Investigator: Volker, Louise, Katrin, Leonie, Flo

Aim of the experiment: Analytical gelelectrophoresis of QuickChange products of P116 and P109 to check whether the QuickChange PCR and DpnI digestion were successful

Procedure:

• 5 µl of the PCR Quickchange products were either mixed with with 0.5 µl of DNA loading buffer (10x) and analytical gelelectrophoresis was performed at 1% agarose gel for 60 min at 90 V.

Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Investigator: Volker

Aim of the study: Picking of transformed Plasmid E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).

Procedure:

• Picking and overnight culture after standard laboratory's protocol. (CamR LB-medium)

• 5 colonies for P116QC (Laccase, QC with O28/O29) and 6 colonies for P109QC (EreB, QC with O18/O19) were picked from the plates.

Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Investigator: Katrin, Louise

Aim of the experiment: Miniprep of overnight cultures of transformated E. coli XL1 blue with QuickChange products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19).

Procedure:

• Miniprep was performed after manufacturer's protocol (QIAprep Miniprep, QIAGEN)

Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19) were successful

Investigator: Katrin, Louise

Aim of the experiment:Analytical digestion and gelelectrophoresis to check whether the transformation of E. coli Xl1 Blue with QuickChanges products of P116 (Laccase, QC with O28/O29) and P109 (EreB, QC with O18/O19)

Procedure:

• analytical digestion of Quickchange products of P109 (EreB, QC with O18/O19)

• in order to check if the QC was successful, P109 (before QC) was also digested

• analytical digestion of Quickchange products of P116 (Laccase, QC with O28/O29)

• in order to check if the QC was successful, P116 (before QC) was also digested

• Incubation for 90 min at 37 °C.

• Analytical gelelectrophoresis was performed at 90 V for 60 min.

Laccase P124 was chosen for sequencing

EreB P125 was chosen for sequencing

Preparative digestion and gelelectrophoresis of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI

Investigator: Andi

Aim of the experiment:Preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeI, P39 with AgeI & PstI, P20 with NgoMIV & PstI to clone fragment of P45 into F17 (pSB1C3) and to fusion Middle linker of P39 with GFP of P20.

Procedure:

Batch for preparative digestion of P45 (FluA-Psb1C3) with XbaI & AgeIwith XbaI & AgeI.

Batch for preparative digestion of P39 (Middle-Linker) with PstI and AgeI.

Batch for preparative digestion of P20 (GFP-Psb1C3) with NgoMIV and PstI.

• Incubation for 120 min at 37 °C.

• Preparative gelelectrophoresis was performed at 90 V for 1.5 h.

• Bands were extracted by QIAquick Gel Extraction Kit, QIAGEN.

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P124(Laccase), P125 (EreB)

Investigator: Andi

Aim of the experiment: Removal of forbidden restiction sites from P124 (Laccase), P125 (EreB).

Procedure: Quickchange - PCR

Reaction batch - P124

Reaction batch - P125

PCR cycling parameters for both batches

• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3)

Investigator: Jeff

Aim of the experiment: Ligation of F29+F30 (GFP in pSB1C3 after middle linker, ( Gly-Gly-Ser-Gly)x2) and F17+F27 (FluA in pSB1C3).

Procedure:

• Ligation batch for F29+F30:

• Ligation batch for F17+F28:

• Negative control was also prepared. Instead of the insert, the same volume of ddH₂O was added to the reaction batch.

• Cycled ligation has been performed after following protocol in a thermocycler:

• Lid temperature = 37 °C

Sequencing of P133 and P134

Investigators: Louise, Johanna

Aim of the experiment: Sequencing of two more QCII products (EreB) Procedure: The DNA was prepared for sequencing according to the Eurofins SmartSeq Kit protocol (15 µl of plasmid DNA (50 - 100 ng) and 2 µl sequencing primer).

The different genes we sequenced received the following barcodes:

Quick Change mutagenesis to remove forbidden restriction sites - SDMIII - of P133 (EreB)

Investigator: Louise

Aim of the experiment: Removal of forbidden restiction site from P133 (EreB).

Procedure: Quickchange - PCR

Reaction batch

PCR cycling parameters - P133

• Digestion of the parental DNA with DpnI: Addition of 1 µl DpnI to the reaction batch and incubate for 1 h at 37 °C.

Transformation of the Quickchange product into E. coli XL1 blue

Investigator: Louise

Aim of the experiment: Transformation of E. coli XL1 blue with Quickchangeproduct SDM III of P133.

Procedure:

• CaCl2 competent E. coli XL1-Blue cells were put out from the stock in -80 °C freezer and were gently thawed on ice.

• 10 µl of DNA was added to 100 µl of competent cells and gently mixed.

• 30 min incubation on ice

• 5 min. heat shock at 37 °C

• Adding of 1 ml LB-medium.

• Incubation for 45 min at 37 °C in the 180 rpm cell-culture shaker.

• 100 µl of the cell suspension was plated on one chloramphenicol plate.

• The rest were centrifuged for 1 min at 13000 rpm and the supernatant was dicarded.

• The pellet was resuspended in 100 µl of LB-medium and this concentrated cell suspension was plated again on a new chlorampenicol plate.