iGEM 2017 — Team Stuttgart

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# Team:Stuttgart/Notebook

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    Information](https://2017.igem.org/Team%3AStuttgart/Background-Information)
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  + [Notebook](https://2017.igem.org/Team%3AStuttgart/Notebook)
  + [Interlab Study](https://2017.igem.org/Team%3AStuttgart/InterLab)
  + [Modelling](https://2017.igem.org/Team%3AStuttgart/Model)
  + [Composite Part](https://2017.igem.org/Team%3AStuttgart/Composite%20Part)
  + [Safety](https://2017.igem.org/Team%3AStuttgart/Safety)
* Results
  + [Esterases and Lipases](https://2017.igem.org/Team%3AStuttgart/Esterases%20and%20Lipases)
  + [Keratinases](https://2017.igem.org/Team%3AStuttgart/Keratinases)
  + [Rose and Limonene Fragrance](https://2017.igem.org/Team%3AStuttgart/Rose%20and%20Limonene%20Fragrance)
  + [Achievements](https://2017.igem.org/Team%3AStuttgart/Demonstrate)
* Human Practices
  + [Public Engagement](https://2017.igem.org/Team%3AStuttgart/Engagement)
  + [Integrated and Gold](https://2017.igem.org/Team%3AStuttgart/HP/Gold_Integrated)
  + [Silver HP](https://2017.igem.org/Team%3AStuttgart/HP/Silver)
  + [Collaborations](https://2017.igem.org/Team%3AStuttgart/Collaborations)
* Awards
  + [Applied Design](https://2017.igem.org/Team%3AStuttgart/Applied_Design)
  + [Measurement](https://2017.igem.org/Team%3AStuttgart/Measurement)
* [Judging Form](https://igem.org/2017_Judging_Form?team=Stuttgart)

# Notebook

## Esterases and Lipases

![](https://static.igem.org/mediawiki/2017/a/ad/Lipaseecomicneu234.jpeg)

03.08.2017

* Transformation of [BBa\_K1149002](http://parts.igem.org/Part%3ABBa_K1149002) and [BBa\_K1149003](http://parts.igem.org/Part%3ABBa_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](http://parts.igem.org/Part%3ABBa_K1149002) and [BBa\_K1149003](http://parts.igem.org/Part%3ABBa_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](http://parts.igem.org/Part%3ABBa_K1149003) and BBa\_K1149002)
* SOC media preparation
* Transformation pet19-LipB

21.08.17

* Preparation: preliminary test of esterase assay with EstCS2: [BBa\_K1149002](http://parts.igem.org/Part%3ABBa_K1149002) - preparation of o/n cultures (37°C)

22.08.17

* Preparation: preliminary test of esterase assay with EstCS2: [BBa\_K1149002](http://parts.igem.org/Part%3ABBa_K1149002) - glycerol stocks and induction with arabinose

23.08.17

* Preliminary test of esterase assay with EstCS2: [BBa\_K1149002](http://parts.igem.org/Part%3ABBa_K1149002) (link results)

![](https://static.igem.org/mediawiki/2017/7/74/Labimpressions3.jpeg)

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

![](https://static.igem.org/mediawiki/2017/5/59/LipB2.jpeg)

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](http://parts.igem.org/Part%3ABBa_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 DH5alpha *E. coli* cells

![](https://static.igem.org/mediawiki/2017/5/5f/Labimpressions4.jpeg)

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 DH5alpha *E. coli* cells

19.09.17 – 21.09.17

* InterLab study LUDOX measurement
* Enzyme activity assay with [BBa\_K1149002](http://parts.igem.org/Part%3ABBa_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

![](https://static.igem.org/mediawiki/2017/c/cb/Labimpressions5.jpeg)

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

![](https://static.igem.org/mediawiki/2017/4/40/Lipa6assay.jpeg)

19.10.17

* PCR - amplification of LipB - cloning of LipB in the iGEM standard plasmid ([BBa\_K1149002](http://parts.igem.org/Part%3ABBa_K1149002) is used)
* PCR - amplification of [BBa\_K1149002](http://parts.igem.org/Part%3ABBa_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](http://parts.igem.org/Part%3ABBa_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

![](https://static.igem.org/mediawiki/2017/3/3c/Lipaseassay.jpeg)

## Keratinases

![](https://static.igem.org/mediawiki/2017/2/21/Keratinasercomicneu234.jpeg)

26.07.17

* Transformation of kerUS ([BBa\_K1498000](http://parts.igem.org/Part%3ABBa_K11498000))
* 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](http://parts.igem.org/Part%3ABBa_K11498000))) 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\_K149002](http://parts.igem.org/Part%3ABBa_K1149002) and [BBa\_K149003](http://parts.igem.org/Part%3ABBa_K1149003)) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C
* Transfer of [BBa\_K1498000](http://parts.igem.org/Part%3ABBa_K11498000) 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

![](https://static.igem.org/mediawiki/2017/0/0d/Labimpressions6.jpeg)

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

To add our keratinase genes with promotors we transformed BBa\_J23115, BBa\_J23119 and an inducible pBAD promotor BBa\_K206000 and cut cleaned up plasmids with EcoR1 and Spe1 for Standard Assembly.
After digest with second enzyme, for J23115 and J23119 only plasmid backbones were detected.
In lines of K206000 two fragments were detected, as expected (insert and backbone) (figure ).

[![](https://static.igem.org/mediawiki/2017/6/6f/Notebookgeldigestpromotors1.png)](https://static.igem.org/mediawiki/2017/6/6f/Notebookgeldigestpromotors1.png)

[![](/wiki/skins/common/images/magnify-clip.png)](https://static.igem.org/mediawiki/2017/6/6f/Notebookgeldigestpromotors1.png "Enlarge")

**Figure 1:** Agarose-gel electrophoresis of ligation products.

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

![](https://static.igem.org/mediawiki/2017/f/f9/Keratinasesd67.jpeg)

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

[![](https://static.igem.org/mediawiki/2017/1/11/Bild10georg.png)](https://static.igem.org/mediawiki/2017/1/11/Bild10georg.png)

[![](https://static.igem.org/mediawiki/2017/2/29/Digest_of_overlapping_PCR_products.png)](https://static.igem.org/mediawiki/2017/2/29/Digest_of_overlapping_PCR_products.png "Enlarge")

**Figure 2:** Digest of kerP plasmids.

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

![](https://static.igem.org/mediawiki/2017/8/8a/Labimpressions2.jpeg)

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

![](https://static.igem.org/mediawiki/2017/8/87/Stuttgart_compis.jpeg)

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

[![](https://static.igem.org/mediawiki/2017/3/30/Gelegeorgneu324.png)](https://static.igem.org/mediawiki/2017/3/30/Gelegeorgneu324.png)

[![](/wiki/skins/common/images/magnify-clip.png)](https://static.igem.org/mediawiki/2017/3/30/Gelegeorgneu324.png "Enlarge")

**Figure 3:**

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

![](https://static.igem.org/mediawiki/2017/d/d2/Azokeratin.jpeg)

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

![](https://static.igem.org/mediawiki/2017/6/63/Keratinasehair1.jpeg)

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)

![](https://static.igem.org/mediawiki/2017/3/30/Keratinaseassay5.jpeg)

## Rose and Limonene Fragrance

![](https://static.igem.org/mediawiki/2017/5/54/Rosenduftcomicneu2343.jpeg)

12.07.17

* Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (pET28a-KDC-YjgB-ARO8: 351,9 ng/µl and pET28a-ATF1: 352,5 ng/µl)
* Preparation of Kanamycin stocks (50µg/ml)

14.07.2017

* Preparation of LB-Agar plates with kanamycin (50 µg/mL)

28.08.17

* Preparation of LB-Agar plates with kanamycin (50 µg/mL)
* Sequencing of rose fragrance plasmids from Guo et al. (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1)

13.09.17

* Overlap-PCR of 1.pBAD (BBa\_K206000), 2.KDC-YjgB-ARO8, 3.ATF1 and 4.pBAD-KDC-YjgB-ARO8
* PCR-Purification and agarose-gel-electrophoresis of PCR products, PCR 4 (pBad-KDC-YjgB-ARO8) not sucessfull

* Measurment of DNA concentration with Nano-Drop:

1. KDC-YjgB-ARO8:54,4 ng/µl
2. ATF1:57,4 ng/µl
3. pBAD:140,7 ng/µL

![](https://static.igem.org/mediawiki/2017/9/91/Nicikerateam1.jpeg)

14.09.17

* Preparation PCR Lemonen and Agarose-Gel

19.09.17

* Preparation of LB-Agar plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL)
* Repeat of PCR 4 (pBAD-KDC-YjgB-ARO8)
* PCR-Purification and agarose-gel-electrophoresis of PCR product 4

* PCR 4 (pBAD-KDC-YjgB-ARO8) not successful
* Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C

21.09.17

* Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1 in psB1K3 backbone

* Gibson Calculater:

1. pSB1K3: 0,55 µL
2. pBAD: 0,74 µL
3. KDC-YjgB-ARO8: 1,91 µL
4. ATF1: 1,81 µL

* Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C

![](https://static.igem.org/mediawiki/2017/0/05/Labimpressions19.jpeg)

22.09.17

* Rose-plasmid Transformation successful
* Single colonies are plated on agar plates and incubated at 37 °C over night

25.09.17

* Verification of transformed rose-plasmid by colony-PCR
* Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful

26.09.17

* Repeat of colony-PCR with transformed rose-plasmid
* Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful

27.09.17

* Repeat of colony-PCR with transformed rose-plasmid
* Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful

28.09.17

* Mini-prep of transformed rose-plasmid

* Nanodrop Measurement:

1. Colony 1: 192,1 ng/µL
2. Colony 2: 159,8 ng/µL
3. Colony 3: 159,1 ng/µL
4. Colony 4: 100,1 ng/µL
5. Colony 5: 118,2 ng/µL

* Restriction assay of isolated rose-plasmid, cut with SpeI
* Verification of restriction product by agarose-gel-electrophoresis - not successful

![](https://static.igem.org/mediawiki/2017/c/c3/AnnaLenasitzt2.jpeg)

29.09.17

* Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1
* Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C - transformation not successful

05.10.17

* Repeat Restriction assay of isolated rose-plasmid, cut with EcoRI - not successful
* Verification of restriction product by agarose-gel-electrophoresis - not successful

12.10.17

* Gibson Assembly of limonene PCR-products () in psB1C3 backbone
* Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C - transformation not successful

17.10.17

* Gibson-Assembly:

1. PSB1C3: 1,75 µl
2. F1: 1,36 µl
3. F2: 1,89 µl

![](https://static.igem.org/mediawiki/2017/a/a0/Limonenepathway.jpeg)

18.10.17

* Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C
* Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful
* M9 media preparation

20.10.17

* Repeat overlap-PCR of pBad and KDC-YjgB-ARO8
* Verification of PCR products by agarose-gel-electrophoresis

23.10.17

* Sequencing of transformed rose fragrance plasmid (pSB1K3-pBad-KDC-YjgB-ARO8-ATF1)
* Sequencing of transformed limonene fragrance plasmid (pSB1C3-pBad)

25.10.17

* Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1, plasmid backbones pSB1K3 and pSB1C3
* Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL) at 37°C - transformation not successful

26.10.17

* Repeat transfomation of rose-plasmid in competent NEB-cells and and over-night incubation on agar-plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL) at 37°C - transformation successful

27.10.17

* Verification of transformed rose-plasmid by colony-PCR
* Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful


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# Team:Stuttgart/Experiments

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# Experiments

PART I - ESTERASES and LIPASES  ![](https://static.igem.org/mediawiki/2017/d/dc/Esterase.png) |

PART II - KERATINASES    ![](https://static.igem.org/mediawiki/2017/6/65/Keratinase.png)Semi-quantitative hair degradation assay To prove any enzyme activity a semi-quantitative hair degradation assay was performed. First, cultures of E. coli containing kerA, kerUS, kerP plasmid and one wild-type E. coli were grown at 37°C in sterile LB broth. Chloramphenicol (final concentration 35 µg/mL) was added to the cultures containing kerA and kerUS. Kanamycin (final concentration 50 µg/mL) was added to the cultures containing the kerP. After incubation OD600 was measured before inducing cultures containing kerA and kerUS with IPTG with a final concentration of 1mM. Human hair was reduced to smaller pieces and then dried for 1 hour at 65°C. Afterwards the hair was distributed in 0.05 g aliquots and the full amount of each culture was added. The cultures + hair were incubated 120 hours at 37°C with slightly shaking.   Skim milk plate assay This assay was performed to show qualitative enzyme activity. The different keratinases (kerA, kerUS and kerP) should degrade the casein in the milk, seen as clear zones around the cultures itself or the supernatant of the cells.   1. Preparation of skim milk plates         First 100 mL LB/agar (see [protocols](https://2017.igem.org/Team%3AStuttgart/Protocols)) and 5 g skim milk powder in 125 mL distilled water was prepared. Both solutions were autoclaved and mixed after cooling down. For the plates used for kerA and kerUS cultures chloramphenicol (35 µg/mL) was added – in case of kerP kanamycin (50 µg/mL) was used to prepare plates. The plates were poured and stored at 4°C.  2. Assay         kerA/kerUS: Before spreading both keratinases on the plates, cells were induced with 1mM IPTG o/n at 37°C. After incubation 10 µL of the induced cell cultures and supernatant, cell culture without IPTG induction and a wild-type E. coli control was spread on skim milk plates containing chloramphenicol. The plates were incubated at room temperature for four days.         kerP: 10 µL of cell culture, supernatant and a wild-type E. coli control was spread on skim milk plates containing kanamycin. The plates were incubated at room temperature for four days. |

PART III - A LOVELY SCENT OF...   ![](https://static.igem.org/mediawiki/2017/d/d3/Rosenduftcomic.png)...ROSE FRAGRANCE  ...LIMONENE FRAGRANCE |

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# Team:Stuttgart/Results

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# Results

### Esterases and Lipases

![](https://static.igem.org/mediawiki/2017/7/7f/EsteraseundLipase.png)

#### Enzyme activity assay: Esterases

In the following figures enzyme activities of the supernatant of the esterases EstCS2 and LIpB are shown. All results were obtained from biological triplicates. The detailed method for the activity determination is shown here: LINK
At the N-terminal end of the esterase EstCS2 a signal peptide (PelB) is added. Therefore the gene induction leads to enzyme expression and the expression of the signalpeptide and the secretion of the enzyme is enabled.

![](https://static.igem.org/mediawiki/2017/4/45/EsteraseResults.png)

The enzyme activity of EstCS2 raises with the induction level for gene expression and with the substrate concentration. A maximum of 246,5 U is gained with 20 mM p-Nitrophenyl butyrate and 1 mM arabinose. The enzyme activities between the induction levels of 2 mM arabinose and 3 mM arabinose isn’t different. The enzyme activity is highest with an induction level of 1 mM arabinose instead of an induction level of 2 or 3 mM arabinose. This could be explained with less movement flexibility of the esterases if high gene expression rates are induced. We would further recommend to induce the gene expression with less arabinose concentrations and investigate the enzyme activity with 0.1 mM, 0.5 mM and 1 mM arabinose.

![](https://static.igem.org/mediawiki/2017/a/a0/EsteraseResults2.png)

###### Figure 4: Illustration of the esterase activity [U] of E. coli wilde types in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.

In comparison we investigated the enzyme activity of two E. coli wilde type strains (E. coli DH5α and MG1655). We aimed to aquire knowledge about the esterase activity of the wild typs to identify the effect of genetically engineered organisms. The figures show less enzyme activity of the wild types in comparison to the genetically engineered E. coli that contain the plasmid with the esterase genes. This means that the investigated EstCS2 is an appropriate enzyme to degradade fat layers on hair.

![](https://static.igem.org/mediawiki/2017/3/3a/EsteraseResults3.png)

###### Figure 5:Illustration of the esterase activity [U] of LipB in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.

Additionally we investigated the enzyme activity of LipB. To compare the enzyme activities of LipB and EstCS2, we used the same induction levels and substrate concentrations for the assays. The figures show that the enzyme activity of the supernatant isn’t higher than the enzyme activity of the supernatant of the wild type cells. These results can be declared with the absence of a signal peptide at the N-terminal side of the esterase gene. Thus, no enzyme secretion is performed and less enzyme activity can be detected in the supernatant. This leads to the conclusion that a signal peptide has to be added at the N-terminal side of the esterase gene to obtain enzyme secretion and extracellular enzyme activity.

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