Team:Stuttgart/Notebook

Notebook

Esterases and Lipases

03.08.2017

• Transformation of BBa_K1149002 and BBa_K1149003

04.08.17

• Single colonies (BBa_K1149002 and BBa_K1149003) are plated on agar plates and incubated at 37 °C over night

09.08.17

• Growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C

10.08.17

• Preparation of miniprep (Jena Biosciences Kit) and glycerol stock (storage: -80 °C) (BBa_K1149002 and BBa_K1149003)
• SOC media preparation
• Transformation pet19-LipB

21.08.17

• Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - preparation of o/n cultures (37°C)

22.08.17

• Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - glycerol stocks and induction with arabinose

23.08.17

• Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results)

24.08.17

• Transformation of LipB into Zymo Research competent mix and go cells
• Preparation of LB medium and new agar plates

25.08.17

• Single colonies of LipB are plated on agar plates and incubated at 37 °C over night

28.08.17

• Preparation of electro-competent cells
• Growing of a single colony (LipB) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C

31.08.17

• Transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)

01.09.17

• Transformation/electroporation with pUC19 plasmid (o/n incubation, 37°C - transformation failed)

06.09-08.09.17

• Enzyme activity assay (cell pellet and supernatant) with BBa_K1149002 and pet19-LipB

12.09.17

• Preparation of chemo-competent cells

13.09.17

• Transformation with iGEM competent cells test kit DNA and pUC19 plasmid (o/n incubation, 37°C) - transformation successful

14.09.17

• Calculation - efficiency of the chemo-competent cells/pUC19: efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL

18.09.17

• Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells

14.09.17

• Calculation - efficiency of the chemo-competent cells/pUC19: efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL

18.09.17

• Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells

19.09.17 – 21.09.17

• InterLab study LUDOX measurement
• Enzyme activity assay with BBa_K1149002 of the supernatant - measurement in biological triplicates
• InterLab study Fluorescein measurement

19.09.17

• Preparation PCR Purification Lipase:

1. prtE-f4: 1410 bp
2. prtF_f5: 1691 bp
3. LARD_f2: 683 bp
4. prtD_f3: 683 bp
5. LARD_f2: 683 bp
6. pBAD_f1: 1660 bp

22.09.17

• InterLab study sample measurement (link results)

27.09.17 - 29.09.17

• Enzyme activity assay with pet19-LipB of the supernatant - measurement in biological triplicates

06.10.17

• Collaboration iGEM team Heidelberg: preparation mutagenesis plasmid activity assay - preparation of media, antibiotic-stocks and sugar-stocks + agar plates with different antibiotics

10.10.17

• Collaboration iGEM team Heidelberg: preparation of different cultures + induction of the cells with arabinose (o/n incubation, 37°C)

11.10.17

• Collaboration iGEM team Heidelberg: spread the o/n cultures on prepared agar plates (o/n incubation, 37°C)

12.10.17

• Gibson Assembly of LipB in psB1C3 backbone

19.10.17

• PCR - amplification of LipB - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)
• PCR - amplification of BBa_K1149002 (without EstCS2)

20.10.17

• SDS page, Gibson Assembly, Transformation (Zymos) - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)

23.10.17

• 5x pelB LipB with 5ml LB over night at 37°C

24.10.17

• Miniprep (Jena Bioscience Kit)
• Induction of enzyme expression for the enzyme activity assay for the Gibson Assembly (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)
• Sequencing of the Gibson Assembly (PelB-LipB)

25.10.17

• Enzyme activity assay (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)

26.10.17

• OD600 determination of Lipase TliA

27.10.17

• Colony PCR of the Gibson Assembly product (PelB-LipB), SDS-PAGE

Keratinases

26.07.17

• Transformation of kerUS (BBa_K1498000)
• Preparation of antibiotic stock solutions: chloramphenicol (35 mg/mL) and ampicillin (100 mg/mL)

27.07.17

• Growing of a single colony (kerUS (BBa_K1498000)) in 5 mL LB media chloramphenicol (35 µg/mL) at 37 °C
• Single colonies are plated on agar plates and incubated at 37 °C over night
• Growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C
• Transfer of BBa_K1498000 into glycerin culture and storage at -80° freezer

28.07.17

• Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))

16.08.17

• Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000

17.08.17

• Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again, after nothing grew on the agar plates
• Competent cell test - not succesfull

22.08.17

• Preparing LB (5ml tubes) and (chemical) competent cells (link prtokoll openwetware)

23.08.17

• preparing primer for overlapping PCR to get restriction sides to kerP: -> preparing and dilute, following IDT protocols.

PCR-cycler conditions:

Step	Cycles	Temperature	Time
Denaturation		98°C	30 sec
Annealing	35	68°C	30 sec
Elongation		72°C	30 sec
final extension	1	72°C	2 min
hold		4-10°C

PCR-Purification with JenaBioScience-kit

24.08.17

• Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3
• Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again into Zymo Research Competent mix and go cells this time, after nothing grew on the agar plates

25.08.17

• New transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again into Zymo Research Competent mix and go on new agar plates from 24.8.17

28.08.17

• Preparation of electro-competent cells
• Preparation of electro-competent cells
• Preparation of new agar plates with Ampicilline and Chloramphenicole
• preculturing E.coli for electroporethic competent cell assay
• Single colonies of promotor (BB_K206000) are plated on agar plates and incubated at 37 °C over night ( other promotors didn’t grow again)
• Transformation of KerA and KerUS on pSB1C3 vector from Canada into Zymo competent mix and go cells (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173)

29.08.17

• Single colonies of (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173) are plated on agar plates and incubated at 37 °C
• Growing of a single colony (BBa_K206000) in 5 mL LB media + ampicilline (100 µg/mL) at 37°C

30.08.17

• Transformation of psB1K3-KerP
• Repeat Ligation with another backbone: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3
• Transformation of three other promotor (BBa_J23100, BBa_J23119, BBa_J23119) and one RBS (BBa_B0034) into Zymo Competent Mix and Go cells
• Preparation Mini-Prep and glycerol storage at -80°C freezer of promotor (BBa_K206000): final concentration: 77,8 ng/µl
• Growing of a single colony (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C

31.08.17

• Transformation of Ligation-product kerP_pSB1K3 with electroporation preparing competent cells (protocol igem)
• Preparation Mini-Prep and glycerol storage at -80°C freezer of (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173): final concentration: BB-K1717000: 305 ng/µl, BBa_K1717173: 232,4 ng/µl

01.09.17

• Mini-prep (jenabioscience kit): BBa_23119, BBa_23115, RBS BBa_B0034

04.09.17

• Transformation of BBa_J04450 -> making pSB1K3 backbone

05.09.17

• Colony-PCR with colony kerP_pSB1K3
• Transformation of two signal peptides: pelB: BBa_K208004 and OmpA: BBa_ K103006 in Zymo Competent mix and go cells

06.09.17

• Transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go
• Single colonies of signal peptides BBa_K208004 and BBa_ K103006 are plated on agar plates and incubated at 37 °C

07.09.17

• Primer-Design and ordering
• Growing of a single colony (BBa_K208004 and BBa_ K103006) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C
• Prepration of LB medium and agar plates

08.09.17

• Preparation Mini-Prep and glycerol storage at -80°C freezer of signal peptides (BBa_K208004 and BBa_ K103006): BBa_K208004: final concentration: 268,7 ng/µl and BBa_K103006: 187,9 ng/µl

11.09.17

• pSB1K3_kerP + 5ml LB over night at 37°C, BBa-J04450 + 5ml LB over night at 37°C

13.09.17

• Mini-prep kerP and BBa_J04450

15.09.17

• Overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB

PCR-cycler conditions:

Step	Cycles	Temperature	Time
Denaturation		98°C	30 sec
Annealing	35	68°C	30 sec
Elongation		72°C	2 min
final extension	1	72°C	2 min
hold		4-10°C

18.09.17

• Agarose gel with PCR products, repeat overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB split of for better range of annealing temperature (65°C)
• Preparation PCR for KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173

19.09.17

• Preparation PCR Purification:

1. OA-A: 73,4 ng/µl
2. KA-OA:90,4ng/µl
3. KA-PB: 77,4 ng/µl
4. PB-A: 22,8 ng/µl
5. KUS-PB: 110,8 ng/µl
6. PB/US: 13,9 ng/µl
7. KUS-OA: 81,9 ng/µl
8. OA-US: 99.5 ng/µl

• Gibson Calculater:

1. Vektor: 163 ng/µl
2. KerA+OmpA: 173 ng/µl
3. OA-A: 162 ng/µl
4. Kerus-OmpA, Vektor: 176 ng/µl
5. KUS-OA: 188 ng/µl
6. OA-US: 175 ng/µl

• Transformation into Zymo competent mix and go cells afterwards.

06.10.17

• Preparation of Keratinase-Assay. Due to troubles to dilute Azo-Keratine, Assay was not successful and has to be repeated.
• Preparation of Azo-Keratine (milling) in Tris/HCL Buffer (pH 8) (2,94 g Tris into 500 ml HCL Buffer)

10.10.17

• Keratinase assay -> preparing substrate azure keratin, problems with insoluble substrate

12.10.17

• Gibson Assembly KerA-ompA, ompA-KerA, KerUS-ompA and ompA-KerUS in psB1C3 backbone

16.10.17

• Preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)
• Preparation skim-milk-plate assay: preparation of skim-milk-agar-plates with chloramphenicol/kanamycin

17.10.17

• Loading sceme on gel for restriction digest for kerA and KerUS M-KerA-KerUS-BBa_J23115-BBa_J23119_BBa_K206000

18.10.17

• Preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)
• Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)
• Preparation of miniprep (Jena Bioscience), standard assembly (used restriction enzymes: EcoRI, XbaI, SpeI) and transformation of: KerA-OmpA and KerUS-OmpA with each of them combined to three promotors: BBa_J23119, BBa_J23115, BBa_K206000.

19.10.17

• Semi-quantitative keratinase-assay: add 0.005 g of dried hair (65 °C, 1h) to o/n cultures (KerUS and KerA are induced with 1mM IPTG before) - 5 days incubation, 37°C (link results)
• Skim-milk-plate assay kerP - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)

20.10.17

• Skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)
• Sequencing of keratinase plasmids: pSB1C3-KerA-ompA, pSB1C3-KerUS-ompA, pSB1C3-KerA_Kanada and pSB1C3-KerUS-Kanada

24.10.17

• Ligation with kerP digest and pSB1C3 backbone

25.10.17

• Analysis of Sequencing (GATC) of different fragments:

1. KerA-OmpA: FW: 1057 bp, RV: 772 bp
2. KerUS-OmpA: FW: 1048 bp, RV: 789 bp
3. KerA (BBa_K1717000): FW: 1107 bp, RV: 837 bp
4. KeratinaseUS (BBa_K1717173): FW: 1148 bp

Measurment of DNA concentration with Nano-Drop:

1. Biobasic KerUS pet28b+:30,3 ng/µl
2. Biobasic KerA pet28b+:74,0 ng/µl
3. KerUS pet28b+:1286,7 ng/µl
4. KerA pet28b+:2465,4 ng/µl
5. KerUS pSB1C3:1588,2 ng/µl
6. KerA pSB1C3:1826,2 ng/µl

• GATC Sequencing of:

1. KerP pet28b+
2. KerUS pSB1C3
3. KerA pSB1C3
4. KerA pet28b+
5. KerUS pet28b+

26.10.17

• Preparation of Lipase Assay with 0M and 3M induction of IPTG and five different substrate concentrations: 2,5; 5; 10 ; 15; 20 µg/ml

30.10.17

• Cell Lysis of different Keratinases strains (look it up from the days before)