Team:TU Darmstadt/Labjournal/Degradation

From 2012.igem.org

Degradation

This page features the work carried out by the degradation team. The main objectives were the production of a BioBrick containing Fusarium solani cutinase or Est13 esterase two enzymes potentially enabling E.coli of PET degradation and over-expression stems for activity screening.

The degradation group consists of six undergraduates and two PhD student advisors. Our objective is the expression of a fusion protein on the surface of E. coli to enable a microbial polyethylenterephtalate (PET) degradation.

We identified three potential PET degradation enzymes from literature. Two of them are cutinases HiC (Humicola insolens cutinase) and FsC (Fusarium solani cutinase), the other namely pNB-Est13 beeing an esterase. After a short examination we dropped the HiC due to a temperature optimum of 80+°C. Shortly after the FsC was dropped as well, due to its toxicity for E.coli.
Enzymatical degradation of polyethylen terephtalate (PET) to terephtalic acid (TPA)

In order to maximise the activity we decided to anchor and display the cutinase/esterase directly on the surface of the producing bacterial cell. Surface-exposed enzymes are directly accessible to the respective substrates which no longer have to traverse the cellular membrane barriers. Furthermore, the enzyme reaction occurs in a chemically more defined environment as compared to the interior of a microbial cell. We use the outer membrane proteins of Pseudomonas aeruginosa (EstA) as a membrane anchor and the signaling sequence of PhoA translocators to display the enzyme on the outer surface of E. coli cells. In addition the fusion protein contains a his-tag and a myc-tag for detection via flow cytometry after antibody staining or Western blot.The degradation group consists of six undergraduates and two PhD student advisors. Our objective is the expression of a fusion protein on the surface of E. coli to enable a microbial polyethylenterephtalate (PET) degradation.

At first the signal sequence (PhoA), the catalytic domain (FsC/Est13) and EstA were assembled inline from their respective vectors. This is due to the fact, that by combining multiple parts in the standardized BioBrick vectors, scars with stop codons are generated that would effectively prevent the fusionproteins expression. In consideration of the DNA constructs length we pursued two PCR based synthesis strategies. One being the SKV (standard cloning procedure) the other being the SOE (standard overlap procedure). The first using primers and restriction sites for assembly the latter using overlapping primers. During both assembly procedures restriction sites of PstI, EcorI, SpeI or XbaI were eliminated from the coding sequence by mutagene PCR. In the end we completely changed our assembly strategy, using synthesis products and BsaI sites to put our parts together. After completion the fusionprotein and its subunits were transfered to the BioBrick standard and sent to the registry. For further characterisation the enzymes were overexpressed in E. coli strains Top10, DH5α, Mg1655, screened on tributyrin agar and detected via flow cytometry after performing an antibody staining. The material science group went even further and tried to examine them using AFM.


Contents

SOE PCR

schematic drawing of SOE PCR. the red gene has to be assembled upstream of the blue gene. Pay attention to the primer overlapps.


SOE PCR stands for Splicing by Overlapp Extension PCR. It is a standard overlapp extension procedure, enabling the assembly of genes wihtout performing any cloning, digesting or ligation inbetween. All you need to do ist running PCRs with specific primers. If gene A is the upstream part and gene B has to be assembled downstream of gene A, primer lo of gene A should have an overlapp of around 20 nucleotides complementary to the first 20 nucleotides of gene B. Primer up of gene B should haven a complementary overlapp of 20 nucleotides to the end of gene A.

We stopped using SOE PCR after a couple of weeks. It is a good method to perform mutationial PCR but to assemble genes of the length of BBa_K808032 it is not the best. Due to bad yields of gel extraction after the first assembly steps we stopped SOE PCR and went on with SKV. But eventually we got our chimeric genes synthesized by GeneArt.





Week 1 / CW 17

Tuesday, 24.04.12

Wednesday, 25.04.12

Thursday, 26.04.12

Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE = 25 s

  1. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SKV
    primers: SKV a1 up XbaI & SKV a1 lo NdeI
  2. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Esterase13
    primers: SOE A up & SOE a1 lo
  3. PCR on pEST100]
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with FsC
    primers: SOE A up & SOE a2 lo
  4. PCR on pEST100
    gene of interest: FsC for SOE PCR with Promo-LacO-RBS-Phoa and EstA part1
    primers: SOE b2 up & SOE b2 lo
  5. PCR on pET26b(+)
    gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
    primers: SOE b1 up & SOE Est13 mut lo
  6. PCR on pET26b(+)
    gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
    primers: SOE Est13 mut up & SOE b1 lo

120426 PCR 1-3 dunkel siehe Laborbuch 26.4.tif

  • 2-5 1.PCR, 6-9 2.PCR, 10-13 3.PCR

120426 PCR 4-6 siehe Laborbuch 26.4.tif

  • 2-5 4.PCR, 6-9 5.PCR, 710-13. 6.PCR

Friday, 27.04.12

Week 2 / CW 18

Monday, 30.04.12

  • SOE PCR
    • pNB-Est13 part1 & pNB-Est13 part2, primers: SOE b1 up & SOE b1 lo
    • Promo-LacO-RBS-Phoa & FsC, primers: SOE A up & SOE b2 lo
    • Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE1 = 20s, tE1 = 35s
  • Agarose gel electrophoresis of SOE PCR

120430 SOE PCR1 dunkel siehe Laborbuch 30.4.png

  • SOE PCR of Promo-LacO-RBS-Phoa-FsC worked, pNB-Est13 did not due to missing clean up via Agarose gel electrophoresis, precipitation is insufficient, we do it again
  • PCRs
  1. PCR on pET26b(+)
    gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
    primers: SOE b1 up & SOE Est13 mut lo
  2. PCR on pET26b(+)
    gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
    primers: SOE Est13 mut up & SOE b1 lo
  • Agarose gel electrophoresis
    • 1. PCR worked well 2. PCR did not

120430 PCR -2 der beiden EST Fragmente 1234 est lo 5678 est up.tif

Wednesday, 02.05.12

  1. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
    primers: SOE A up & SOE a1 lo
  2. PCR on pET26b(+)
    gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
    primers: SOE b1 up & SOE Est13 mut lo
  3. PCR on pET26b(+)
    gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
    primers: SOE Est13 mut up & SOE b1 lo
TA = 57°C, ta = 35s, tE = 25 s
every PCR is performed in 3 batches à 50 µL
TA1 = 60°C, ta1 = 25 s, tE1 = 25 s
TA = 60°C, ta2 = 25 s, tE2 = 35 s

Tuesday, 03.05.12

Friday, 04.05.12

  1. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
    primers: SOE A up & SOE a1 lo
  2. PCR on pEST100
    gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13
    primers: SOE c1 up & SOE EstA mut PstI out lo
  3. PCR on pEST100
    gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and FsC
    primers: SOE c2 up & SOE EstA mut PstI out lo
  4. PCR on pEST100
    gene of interest: EstA part2 for SOE PCR with EstA part1
    primers: SOE EstA mut PstI out up & SOE D lo

Week 3 / CW 19

Tuesday 08.05.12

gene of interest: Promo-LacO-RBS-Phoa-pNBEst13 for SOE PCR with EstA
primers: SOE A up & SOE b1 lo
template: pNB-Est13 from last friday & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12
    • annotation: TA1 = 60°C, ta1 = 25 s, tE1 = 35 s
    • TA2 = 60°C, ta2 = 25 s, tE2 = 1 min
  • did not work

Wednesday, 09.05.12

  1. PCR on pET26b(+)
    gene of interest: pNB-Est13 part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13 part2
    primers: SOE b1 up & SOE Est13 mut lo
  2. PCR on pET26b(+)
    gene of interest: pNB-Est13 part2 for SOE PCR with pNB-Est13 part1 and EstA part1
    primers: SOE Est13 mut up & SOE b1 lo
  3. PCR on pEST100
    gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and pNB-Est13
    primers: SOE c1 up & SOE EstA mut PstI out lo
  4. PCR on pEST100
    gene of interest: EstA part1 for SOE PCR with Promo-LacO-RBS-Phoa and FsC
    primers: SOE c2 up & SOE EstA mut PstI out lo
  5. PCR on pEST100
    gene of interest: EstA part2 for SOE PCR with EstA part1
    primers: SOE EstA mut PstI out up & SOE D lo
    • annotation: TA = 60°C, ta = 25 s, tE = 35 s
    • every PCR is performed in 2 batches à 50 µL

Friday, 09.05.12

Week 4 / CW 20

Monday, 14.05.12

  • Gel extraction of remaining PCRs from Wednesday, 09.05.12
    • c(pNB-Est13 part1)=24 ng/µL
    • c(pNB-Est13 part2)=26 ng/µL
    • c(EstA part2)=13 ng/µL
  • new SOE EstA mut PstI out lo is orderd from Sigma-Aldrich
  • SOE PCR
gene of interest: pNB-Est13 for SOE PCR with EstA and Promo-LacO-RBS-Phoa
primers: SOE b1 up & SOE b1 lo
template: pNB-Est13 part1 from last friday & pNB-Est13 part2

Tuesday, 15.05.12

  1. SOE PCR
    gene of interest: Promo-LacO-RBS-Phoa-pNBEst13 for SOE PCR with EstA
    primers: SOE A up & SOE b1 lo
    template: pNB-Est13 from yesterday & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12
  2. SOE PCR
    gene of interest: Promo-LacO-RBS-Phoa-FsC for SOE PCR with EstA
    primers: SOE A up & SOE b2 lo
    template: FsC from Thursday, 26.04.12 & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12
    • annotation: TA1 = 52°C, ta1 = 25 s, tE1 = 35 s
    • TA2 = 57°C, ta2 = 25 s, tE2 = 1.15 min
    • each PCR is performed in 2 batches à 50 µL

Wednesday, 16.05.12

  • SOE PCR of Promo-LacO-RBS-Phoa-pNBEst13 did not work but Promo-LacO-RBS-Phoa-FsC worked
  • Gel extraction
    • c(Promo-LacO-RBS-Phoa-FsC)=10 ng/µL

Week 5 / Kw 21

Monday, 23.05.12

  1. PCR on pEST100
    gene of interest: EstA part1 for SOE PCR with pNB-Est13 and EstA part2
    primers: SOE c1 up & SOE EstA mut PstI out lo
  2. PCR on pEST100
    gene of interest: EstA part1 for SOE PCR with FsC and EstA part2
    primers: SOE c2 up & SOE EstA mut PstI out lo

Tuesday, 22.05.12

Wednesday, 23.05.12

  1. SOE PCR
    gene of interest: EstA for SOE PCR with FsC
    primers: SOE c2 up & SOE D lo
    template: EstA part1 from yesterday & EstA part2 from Monday, 14.05.12
    • annotation: TA1 = 52°C, ta1 = 25 s, tE1 =45 s
    • TA2 = 65°C, ta2 = 25 s, tE2 = 90 s
  • qualitative Agarose gel electrophoresis
  • Gel extraction
    • c(EstA for SOE PCR with pNB-Est13)=42 ng/µL
    • c(EstA for SOE PCR with FsC)=23 ng/µL

Thursday, 24.05.12

  1. PCR on EstA for SOE PCR with pNB-Est13 from yesterday
    gene of interest: EstA for SOE PCR with pNB-Est13
    primers: SOE c1 up & SOE D lo
  2. PCR on EstA for SOE PCR with FsC
    gene of interest: EstA for SOE PCR with FsC
    primers: SOE c2 up & SOE D lo
  3. PCR on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13
    primers: SOE A up & SOE a1 lo
  4. PCR on pEST100
    gene of interest: FsC for SOE PCR with Promo-LacO-RBS-Phoa and EstA part1
    primers: SOE b2 up & SOE b2 lo
    • annotation: TA = 60°C, ta = 25 s, tE =45 s
each PCR is performed in 3 batches à 50 µL
  • qualitative Agarose gel electrophoresis
    • both EstAs worked
    • Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13 worked
  • Gel extraction
    • c(EstA for SOE PCR with pNB-Est13)=39 ng/µL
    • c(EstA for SOE PCR with FsC)=20 ng/µL
    • c(Promo-LacO-RBS-Phoa for SOE PCR with pNB-Est13)=13 ng/µL

Week 6 / Kw 22

Tuesday, 29.05.12

  1. SOE PCR
    gene of interest: Promo-LacO-RBS-Phoa-Fsc-EstA
    primers: SOE A up & SOE D lo
    template: EstA from Thursday, 24.05.12 & Promo-LacO-RBS-Phoa-Fsc from Wednesday, 16.05.12
  2. SOE PCR
    gene of interest: Promo-LacO-RBS-Phoa-pNBEst13-EstA
    primers: SOE A up & SOE D lo
    template: EstA from Thursday, 24.05.12 & Promo-LacO-RBS-Phoa from Wednesday, 02.05.12 & pNB-Est13 from Monday, 14.05.12
  3. SOE PCR
    gene of interest: Promo-LacO-RBS-Phoa-pNBEst13 for SOE PCR with EstA
    primers: SOE A up & SOE b1 lo
    template: Promo-LacO-RBS-Phoa from Wednesday, 02.05.12 & pNB-Est13 from Monday, 14.05.12
    • annotation: each PCR is performed in 3 batches à 50 µL
    • TA1 = 52°C, ta1 = 25 s, tE1 =45 s
    • TA2 = 65°C, ta2 = 25 s, tE2 = 90 s
  • Agarose gel electrophoresis
    • Promo-LacO-RBS-Phoa-Fsc-EstA worked
    • Promo-LacO-RBS-Phoa-pNBEst13 worked

Thursday, 29.05.12

  • Gel extraction
    • c(Promo-LacO-RBS-Phoa-Fsc-EstA)= 8 ng/µL
    • c(Promo-LacO-RBS-Phoa-pNBEst13)= 13 ng/µL



Due to better results in SKV we stopped our tries in SOE PCR assembly of our chimeric protein BBa_K808030.

SKV

Molecular cloning.jpg

The shortcut SKV stands for "Standard Klonierungs Verfahren", in english standard molecular cloning. The team tried to build up the whole construct without usage of the BioBrick system as a second way. Here the team had to generate many primers with different restriction sites to connect the parts of the construct.







Week 1 / CW 17

Tuesday, 24.04.12

Wednesday, 25.04.12

Thursday, 26.04.12

Annotation: Every PCR is done in 4 assays à 50 µL, TA = 60°C, ta = 35s, tE = 25 s

  1. PCR 1on pEST100
    gene of interest: Promo-LacO-RBS-Phoa for SKV
    Primers: SKV a1 up XbaI & SKV a1 lo NdeI

120426 PCR 1-3 siehe Laborbuch 26.4-1.png

  • 2-12= phoA

Friday, 27.04.12

Week 2 / CW 18

Monday, 30.04.12

  • Agarose gel electrophoresis of DNA digestion from Friday, 27.04.12
  • Only one single band on Agarose gel electrophoresis
    • For saveness two single digests are performed at 20 µL batches. One with XbaI and the other with NdeI. Incubation for 2 hours.
  • Agarose gel electrophoresis of single digests
    • Enzymes cut once each, so digest should have worked.

120430 Testrestrikt.XbaINdeI zugeschnitten.png

  • Ligation of PhoA in cut (XbaI / NdeI) pET16b
    • Annotation: 30 µL batch, 1:5 ratio, Ligation at 4°C for 2 days till Wednesday, 02.05.12

Week 4/CW 20

Monday, 07.05.

Annotation: If it does not say anything else, Ligation is always done in 20µl batches.

Tuesday, 08.05.

Wednesday, 09.05.

  • Colony-PCR
    • gene of interest: PhoA
    • primer:SKV a1 up XbaI, SKV a1 lo NdeI
  • PhoA was not inserted, but 2 colonies were picked and inoculated in 5 ml LB Amp medium to look at it again due to a test-restriction.

Thursday, 10.05.

Friday, 11.05.

  • an analytic, 1% agarose-gel proves that the amplification of FSC, Est13 and EstA was successfull.
  • But to amplify Est13 it was used the product of the SOE PCR which had not been cleaned up, so the large Fragments of the SOE-primers might have disturbed the amplification. The PCR of Est13 had to be performed again (Wednesday, 16.05.)

120511 - Kopie1.png

  • 2,3,5,6= Est13
  • 7,8,9,10= EstA
  • 11,12= FSC
  • DNA digestion of pET16b to produce template for a new ligation of PhoA
    • concentration: 76,3 ng/µl
    • enzymes: XbaI, NdeI
    • incubation: for 2 days, 37°C
  • Ligation of PhoA x pET16b cut with XbaI and NdeI is carried out using different rates of Vector and insert:
    • ratio vector/insert= 3:1, 1:1, 1:3, 1:5, 1:7, 1:10
    • PhoA= 10,6 ng/µl Monday,07.05.
    • incubation: 2 days, 4°C

Week5/CW21

Monday, 14.05.

Tuesday, 15.05.

120515 colony PCR + SkV Est13 beschriftet.png

  • PhoA is inserted, so 10 clones (2-9,11,12) are picked to be grown in 5ml DYT over night.

Wednesday, 16.05.

  • Miniprep of 10 clones, the plasmid-DNA is isolated out of 3ml culture per clone.
  • Restriction digest with NdeI and NcoI of the plasmids (phoA x pET16b) 1-4, so FSC and Est13 can be added to the construct.
  • FSC is cut with NdeI and NcoI
  • PCR1 of EST13
    • template: product of SOE-PCR, cleaned up by the Promega-Kit
    • primer: SKV b1 up NdeI, SKV b1 lo NcoI
    • an analytical Agarose gel electrophoresis is performed

120516 SOE PCR PhoA Est13 + SkV Est13 FsC2.png

Friday, 18.05.

  • Ligation of FSC x (phoA x pET16b)
    • ratio vector/insert: 3:1, 1:1, 1:3
    • concentration:
  • (phoA x pET16b)= 3,72 ng/µl
  • FSC= 12,22 ng/µl

Week 6/CW 22

Monday, 21.05.

120523 pET16b Verdau pET16b NN1.png

Tuesday, 22.05.

Wednesday, 23.05.

Thursday, 24.05.

  • Colony-PCR proves, that Est13 has been inserted into (phoA x pET16b)
    • plates are covered with colonies, 8 colonies are chosen for colony-PCR
    • colony No.5 is chosen to be inoculated in 5µ DYT/Amp medium

120524 o Est13 FsC u Kolonie PCR Est131.png

Friday, 25.05.

Week 7/CW 23

Tuesday, 29.05.

  • 120529 EstASKVund SOEXXX1.png

Wednesday, 30.05.

  • Restriction digest of EstA (product of PCR, 80 µl) and Plasmid: (Est13 x phoA x pET16b)
    • enzymes: EcoRI, NcoI
    • incubation: 1,5h, 37°C
    • The Agarose gel electrophoresis shows, that two DNA fragments are cut out of the vector (Est13 x phoA x pET16b). This might be a hint that Est13 is not inserted.

120530 VPE + EstA Reinigung Verdau1.png

    • 1-3 = (Est13 x phoA x pET16b)
    • 4-5 = EstA
    • c(EstA) = 6,7 ng/µl
    • c(Est13 x phoA x pET16b)= 23 ng/µl
  • Electroporation of (FSC x phoA x pET16b) worked well, plates are covered with colonies which are picked to be grown in 5ml DYT/Amp medium over night.

Thursday, 31.05.

  • Miniprep of the amplified (FSC x phoA x pET16b)
  • to be sure that the plasmid (Est13 x phoA x pET16b) really carries Est13 and PhoA, an analytic PCR is performed. Also pEt16b is used as template with different primers.
  • templates:
  • Primers, used on each template:
    • SKV b1 up NdeI, SKV b1 lo NcoI
    • SKV a1 up XbaI, SKV a1 lo NdeI
  • conditions:
    • TA = 66°C, ta = 35s, tE = 90s
  • the Agarose gel electrophoresis shows, that there is no binding-specifity concerning the genes of interest and the primers, but primers have been checked on mistakes.
  • to solve the problem, the Ligation of FSC and Est13 shall be repeated from the beginning.

120531ae Est131.png

  • Additionally,the decision was to go on with the Ligation of EstA into the Plasmids (Est13 x phoA x pET16b) and (FSC x phoA x pET16b), in case that the PCR was proceeded under wrong conditions.

Friday, 01.06.

  • Restriction digest of (phoA x pET16b)
    • enzymes: NcoI, NdeI
    • incubation: 1,5 h, 37°C
  • Ligation of Est13 and FSC x (PhoA x pET16b)
    • ratio vector/insert: 1:3, 1:5
    • c([[Est13)= 3 ng/µl
    • c(FSC)= 11 ng/µl
    • c(phoA x pET16b)= 29 ng/µl
    • incubation: over night, 25°C
  • Restriction digest of (FSC x (PhoA x pET16b)
  • enzymes: NcoI, EcoRI
  • incubation: 1,5h, 37°C

Week 8/CW 24

Monday, 04.06.

Tuesday, 05.06.

Wednesday, 06.06.

  • Electroporation of DH5alpha with Ligation-assays from 01.06.
  • Ligation of EstA Wednesday,30.05 and the Plasmids (Est13 x phoA x pET16b),(FSC x phoA x pET16b) from Monday,04.06.
    • ratio vector/insert: 1:3,1:5
    • incubation: 2h, 25°C
    • Electroporation of DH5alpha with (EstA x Est13 x phoA x pET16b) and (EstA x FSC x phoA x pET16b)

Friday, 08.06.

  • No cells are grown on plates, so the plates are incubated for another two days at RT.

Week8/ CW24

Monday, 11.06.

  • all plates are covered with cells, so a colony-PCR is performed on either Est13,FSC or EstA
  • Primer:
    • SKV b1 up NdeI, SKV b1 lo NcoI
    • SKV b2 up NdeI, SKV b2 lo NCOI
    • SKV c1 up NcoI, SKV c1 lo EcoRI
    • Every PCR is done in 8 assays à 50 µL, TA = 57°C, ta = 35s, tE = 90 s

Tuesday, 12.06.

  • colonies No.3,4 (EstA x FSC x phoA x pET16b) and No.15,16 (EstA x Est13 x phoA x pET16b) seem to be positive on EstA and are picked to be grown in 5ml DYT/Amp medium.
  • Colony-PCR on Est13 and FSC shows a lot of unspecific bands, so cloning of the genes from the beginning has to be dropped.

120612 SKVcolony-PCR,VPFE,VPEE.png

Wednesday, 13.06.

  • Miniprep of the clones No. 3,4,15 and 16. Tuesday, 12.06.
    • c(EstA x FSC x PhoA x pET16b)=
    • No.3=65,17 ng/µl
    • No.4=63,8 ng/µl
    • c(EstA x Est13 x PhoA x pET16b)=
    • No.15=66,3 ng/µl
    • No.16=59,7 ng/µl
  • for gene-expression, it is performed a elektroporation of BL21-cells with 1µl of the PLasmid-DNA which has just been isolated.

Friday, 15.06.

  • Transformation of BL21 worked well
  • Tributyrinagar plates with 1mM IPTG were generated, to induce the Lac-Promotor of pET16b, and to demonstrate the esterase-activity by the lysis of Tributyrin.
  • afterwards Tributyrinagar plates are inoculated with cells which were transformed by Miniprep No.3,4,15,16 on Wednesday, 13.06.
    • incubation: 37°C, 2 days

Week9/CW25

Monday, 18.06.

  • Tributyrinagar plates are covered with cells, but no lysis is visible.
    • plates are incubated for another day at 25°C.

Tuesday, 19.06.

  • no lysis, plates stay incubated at 25°C.

Wednesday, 20.06.

  • no lysis

Friday, 22.06.

  • The plasmids No.3,4 (EstA x FSC x phoA x pET16b) and No.15,16 (EstA x Est13 x phoA x pET16b) are prepared for sequencing.
  • Primer:
  • (EstA x FSC x phoA x pET16b):
    • T7 up
    • T7 lo
    • SOE EstA mut up lo
    • [[SOE EstA mut up lo
  • (EstA x Est13 x phoA x pET16b):
    • T7 up
    • T7 lo
    • SOE Est13 mut up lo
    • SOE Est13 mut up lo

Sequencing was not successfull. It was discovered later, that there was a contamination of the DH5alpha cells with other plasmid-DNA, probably caused by Electroporation. SOE-PCR
Due to this fact, the isolated plasmids were always mixed up with other plasmids, and sequencing had to fail. The contamination of the cells may have also caused false-positive results of the colony-PCRs.

We suggest that the cutinase FSC has an additional lipase-activity, which causes a lysis of the cell-membrane and may have an selective effect on FSC-negative mutants while cultivating the cells.

BioBricks

BioBricks are standardized parts of synthetic biology. For more information visit the BioBrick registry. As a contribution to iGEM it is necessary to submit the isolated enzymes and proteins in the pSB1C3 vector.

Week 1 / CW 21

M Est13 PhoA+RBSx2 PhoAx3 PhoA+RBS Est13x2 FsCx3
Pcr ae 03.JPG
Note: Accidental exchange of PhoA+RBS (57°C) and Est13 (57°C)

Week 2 / CW 22

  • PCR of FsC: 3 assays à 50 µl TAnneal = 57/58,6/60°C @ 120s
Marker FsC x 3
Pcr ae 01.JPG
  • PCR of EstA and Est13: 2 assays à 50 µl TAnneal = 57/62°C @ 120s
M Est13x2 EstAx2
Pcr ae 02.JPG
Note: PCR of Est13 did fail [only primer band]

Week 3 / CW 23

Week 4 / CW 24

Week 5 / CW 25

M PhoA EstA EstA Est13 Est13 FsC Fsc Kol.1 Kol.2
Pcr ae 04.JPG
  • PCR of EstA, Est13 and PhoA (Templates: PhoA = pEst100; EstA,Est13 = komp. Est13 mutated): 3 assays à 50 µl TAnneal = 57/62°C @ 120s
  • 1% Agarose gel electrohporesis and PCR clean-up using the Wizard SV Gel and PCR Clean-Up System

Week 6 / CW 26

  • Cultivation of pSB1C3 in DYT (3ml) @ 37°C
  • Miniprep of the pSB1C3 cutures and determination of the concentration via NanoDrop yields of ~47,02/52,11 ng/ml @ 30 µl each
  • Restriction digest of EstA, Est13, PhoA and pSB1C3 (200 ng each) with PstI & EcoRI (à 20 µl) @ 37°C for 1:55h
  • Heat inactivation of the restriction digest @ 55°C for 25min

Week 7 / CW 27

Gelelectrophoresis of the restriction digest.

M PhoA EstA Est13 - pSB1C3
Pcr ae 05.JPG

Extraction of the PhoA, EstA, Est13 and pSB1C3 from the agarose gel. Ligation and transformation of DH5α.

Week 8 / CW 28

Colony PCR of the cultures.

M PhoA PhoA PhoA PhoA EstA EstA Est13 Est13
Pcr ae 06.JPG

Gel was run too long. Therefore no conclusion in regard of the transformation success was possible.

Week 9 / CW 29

Tuesday, 17.07.12

each PCR is performed in 5 batches à 50 µL.
TA = 57°C, tA = 30 s, tE = 2 min
  1. PCR on pEST100
    gene of interest: PhoA for designing part BBa_K808028
    primers: BBa PhoA up & BBa PhoA down
  2. PCR on pET26b(+)
    gene of interest: pNB-Est13 part1 for designing part BBa_K808026 via SOE PCR
    primers: BBa Est13 up & SOE Est13 mut lo
  3. PCR on pET26b(+)
    gene of interest: pNB-Est13 part2 for designing part BBa_K808026 via SOE PCR
    primers: BBa Est13 down & SOE Est13 mut up
gene of interest: pNB-Est13 for designing part BBa_K808028
primers: BBa Est13 up & BBa Est13 down
    • Annotation: SOE PCR is performed in 5 batches à 50 µL
    • TA1 = 52°C, tA1 = 25 s, tE1 = 75 s
    • TA2 = 57°C, tA2 = 30 s, tE2 = 2 min
    • stored over night at 10°C

Wednesday, 18.07.12

120718 SOE PCR Est13.jpg

gene of interest: EstA from SKV Date for designing part BBa_K808027
primers: BBa EstA up and BBa EstA down
    • Annotation: PCR is performed in 5 batches à 50 µL
    • TA = 57°C, tA = 25 s, tE = 75 s
    • GC Buffer is used
  • preparative Agarose gel electrophoresis
  • Gel extraction of PCR product
    • gene of interest: EstA: c(pNB-Est13 part2)=119 ng/µL
    • stored in freezer

Thursday, 19.07.12

    1. PhoA from Tuesday, 17.07.12
    2. pNB-Est13 fromTuesday, 17.07.12
    3. EstA from Wednesday, 18.07.12
    4. pSB1C3 1
    5. pSB1C3 2

Friday, 20.07.12

Week 10 / CW 30

Monday, 23.07.12

120723 BBaPhoA BBa PhoA BBa FsC BBa FsC BBa Est13 BBa Est13 BBa Est13.jpg

  • 2-3 PhoA colonies, 4-5 FsC colonies, 6-8 pNB-Est13 colonies
  • Miniprep of one inoculated colony in DYT-media with CAM from Friday, 20.07.12
    • c(PhoA in pSB1C3)=220 ng/µL
    • c(pNB-Est13 in pSB1C3)=50 ng/µL
  • inoculation of 2 x 5 mL DYT-media-Cmp with pSB1C3 carrying FsC
  • second transformation of EstA from Friday, 20.07.12 worked, but too many colonies!
    • purity plate is done

Tuesday, 24.07.12

  • Miniprep of the 2 FsC DYT-media-Cmp cultures with pSB1C3 carrying FsC from yesterday
    • c(FsC in pSB1C3)=168 ng/µL
  • purity plate of EstA in pSB1c3 is positive
    • colonies picked
    • Colony PCR with positive control by amplfifying an EstA from SKV
    • Annotation: TA = 57°C, tA = 25 s, tE = 2 min, done with NEB- Phusion so its similiar to PCR 1
    • GC Buffer is used

Wednesday, 25.07.12

120725 Colony BBaEstA positiv probe auf SKV EstA.jpg

  • 2 EstA colony, 4 EstA from SKV as a control
  • Miniprep of inoculated 5 ml DYT-media-Cmp
    • c(EstA in pSB1C3)=350 ng/µL

Thursday, 26.07.12

  • Sequencing is ordered. The following premixes are used :
    • 10 µL of c(PhoA in pSB1C3)=220 ng/µL, 1µL VR, 9 µL ddH2O
    • 10 µL of c(PhoA in pSB1C3)=220 ng/µL, 1µL VF2, 9 µL ddH2O
    • 7 µL of c(FsC in pSB1C3)=168 ng/µL, 1µL VR, 7 µL ddH2O
    • 7 µL of c(FsC in pSB1C3)=168 ng/µL, 1µL VF2, 7 µL ddH2O
    • 5 µL of c(EstA in pSB1C3)=350 ng/µL, 1µL VR, 9 µL ddH2O
    • 5 µL of c(EstA in pSB1C3)=350 ng/µL, 1µL VF2, 9 µL ddH2O
    • 5 µL of c(pNB-Est13 in pSB1C3)=50 ng/µL, 1µL VR, 9 µL ddH2O
    • 5 µL of c(pNB-Est13 in pSB1C3)=50 ng/µL, 1µL VF2, 9 µL ddH2O
  • in order to prepare DMSO stocks and Minipreps 5 mL DYT-media-Cmp are inoculated with following colonies containing
    • pNB-Est13 in pSB1C3
    • PhoA in pSB1C3
    • FsC in pSB1C3
    • EstA in pSB1C3
    • Annotation: incubation is done at 37°C over night

Friday, 27.07.12

  • inoculated pNB-Est13 in pSB1C3 from Thursday, 26.07.12 has not grown over night
    • new colony pcked from plate and incubated in 5 mL DYT-media-Cmp at 37°C over night
  • Miniprep of 3 mL from the following over night DYT-media cultures from Thursday, 26.07.12
    • c(PhoA in pSB1C3)=440 ng/µL
    • c(pNB-Est13 in pSB1C3)=450 ng/µL
    • c(FsC in pSB1C3)=90 ng/µL

Week 11 / CW 31

Monday, 30.07.12

Tuesday, 31.07.12

Trouble shooting

  • Symptoms
    • very much transformants on crossed out plates after bacterial transformation by Electroporation
    • at first Colony PCR is positive
    • Minipreps result in very high yields (exceeding >200 ng/µL)
    • second Colony PCR is negative
    • sequencing failed
    • BUT FsC in pSB1C3
      • has had not very much transformants
      • has had a seuqencing result
      • its Minipreps resulted in medium concentrations, settling around 90 ng/µL
  • Diagnosis:
    • our bacterial transformation is done by Electroporation
    • therefor we use electroporation cuvettes, stored in DNA precipitating liquids like isopropanol or ethanol after being washed out.
    • long before we started these electroporation cuvettes were in use.
    • if not washed out carefully, DNA debris (like vectors and Inserts) accumulates in these storage liquids, due to precipitation
    • when an Electroporation is performed with one of these cuvettes, probably containing only little DNA debris, the cells get transformed with numbers of different precipitated plasmids
    • these plasmids contain a variety of resistances, reaching from CAM over KAN to Amp, allowing even "wrong" transformants to grow on selective media
    • after a short period of time, bacterial colonies start to seperate and repel their plasmids, selecting only the most "comfortables"
    • probably our plasmids, containing big genes on ligated vectors, are pushed out during this phase of selection
    • this slow decrease of plasmid could explain the missing second positive signal of Colony PCR

Wednesday, 01.08.12

  • due to change of strategy the following PCR 1s are performed
  1. PCR on pEST100
    gene of interest: PhoA BioBrick for assembly of part BBa_K808028
    primers: BBa PhoA up & BBa PhoA down
  2. PCR on pEST100
    gene of interest: EstA part1 for SOE PCR of EstA (BBa_K808027)
    primers: BBa Est A up & SOE EstA mut PstI out lo
  3. PCR on pEST100
    gene of interest: EstA part2 for SOE PCR of EstA (BBa_K808027)
    primers: BBa EstA mut up & BBa EstA down
  4. PCR on pET26b(+)
    gene of interest: pNB-Est13 part1 for SOE PCR of pNB-Est13 (BBa_K808026)
    primers: BBa Est13 up & BBa Est13 mut lo
  5. PCR on pET26b(+)
    gene of interest: pNB-Est13 part2 for SOE PCR of pNB-Est13 (BBa_K808026)
    primers: BBa Est13 lo & BBa Est13 mut up
  1. SOE PCR
    gene of interest: EstA
    primers: EstA part1 & EstA part2
  2. SOE PCR
    gene of interest: pNB-Est13
    primers: pNB-Est13 part1 & pNB-Est13 part2

120801 SOE PCR Est13 EstA EstApart2 kontrolle.jpg

    1. PhoA
    2. pNB-Est13
    3. EstA

Thursday, 02.08.12

Friday, 03.08.12

Saturday, 04.08.12

TA = 60°C, ta = 35s, tE = 25 s

120804 Verdau PhoA, EstA, Est13, PCR auf EstA, Est 13, PhoA, Kontrolle auf Insert und Vektor.jpg

Week 12 / CW 32

Assembly of our composite part RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808030) is reached by synthesis (from GENEART) of 2 different gene parts:

Monday, 06.08.12

Tuesday, 07.08.12

Wednesday, 08.08.12

Thursday, 09.08.12

Friday, 10.08.12

  • transformation from yesterday suceeded
    • one colony per plate (1:5:5 & 1:10:10 at room temperature and 1:5:5 & 1:10:10 at room 4°C)is picked for inoculation of 5 mL DYT-media-CAM

Saturday, 11.08.12

120811 PCR (VR,VF2) und Verdau (EcoRI,PstI) auf BBa Gesamt von Geneart.jpg

Week 13 / CW 33

Monday, 13.08.12

  1. PCR 1 on bba prefix-PhoA-His6tag-pNBEst13-BsaI site, as a template the pure synthesis product is used
    TA = 57°C, tA = 25 s, tE = 2 min
    primers: XbaI Rbs Phoa up & Est13 Bsa1 lo
  2. PCR 1 on BsaI-Myctag-EstA- bba suffix, as a template the pure synthesis product is used
    TA = 57°C, tA = 25 s, tE = 2 min
    primers: EstA Bsa1 lo & BBa EstA down
  3. PCR 3 on preped colony (1:5:5 room temperature from Friday, 10.08.12)
    TA = 57°C, tA = 25 s, tE = 2 min
    primers: XbaI Rbs Phoa up & BBa EstA down

120813 PCR 1-4 RBSPE 5-8 EXN-Myc-Est 9-12.jpg

120813 PCR Q5 auf bba gesamt konstrukt.jpg

  1. 1.PCR & 2.PCR in pSB1C3 carrying RFP
  2. 3.PCR in pSB1C3 carrying RFP
  3. 1.PCR & 2. PCR in BBa_K808000 possibility 1
  4. 3.PCR in BBa_K808000 possibility 1
  5. 1.PCR & 2. PCR in BBa_K808000 possibility 2
  6. 3.PCR in BBa_K808000 possibility 2

Tuesday, 14.08.12

Wednesday, 15.08.12

  • transformation BBa_K808000
  • inoculation of 5 mL DYT-media-Cmp of the following colonies
    • colony 1.1
    • colony 2.1
    • colony 2.2
    • colony 3.1
    • colony 4.1
    • colony 4.2
    • colony 5.1
    • colony 5.2
    • colony 6.1
    • colony 6.2
      • incubation over night at 37°C
  • Colony PCR on colonies 1.1-6.2 with house-taq
    • primers: VR & VF2

Thursday 16.08.12

120816 Testrestriktion der pSB1C3 mit e und p colony platten 1 - 6.jpg

  • 2 colony 1.1, 3 colony 2.1, 3 colony 2.2, 4 colony 3.1, 5 colony 4.1, 6 colony 4.2, 7 colony 5.1, 8 colony 5.2
  • for further information see discussion below
  • new inoculation of 5 mL DYT-media-Cmp of Colonies 1.1 - 5.2

Trouble shooting

What should have been transformed:

  • colony 1.1: 1.PCR & 2.PCR in pSB1C3
    • should be RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808030) in pSB1C3 (length: ~ 3.1 kb)
  • colony 2.1: 3.PCR in pSB1C3 upstream of part BBa_K808000 (Arabinose inducible promotor) possibility 1
    • should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808032) in pSB1C3 (length: ~ 4.3kb)
  • colony 2.2: similar to colony 2.1
  • colony 3.1: 3.PCR in pSB1C3 upstream of part BBa_K808000 (Arabinose inducible promotor) possibility 2
    • should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808032) in pSB1C3 (length: ~ 4.3kb) like colony 2.1
  • colony 4.1: 3.PCR in pSB1C3
    • should be RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808030) in pSB1C3 (length: ~ 3.1 kb)
  • colony 4.2: similiar to colony 4.1
  • colony 5.1: 1.PCR & 2.PCR in pSB1C3 upstream of part BBa_K808000 (Arabinose inducible promotor) possibility 1
    • should be Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA (BBa_K808032) in pSB1C3 (length: ~ 4.3kb)
  • colony 5.2: similar to colony 5.1

Conclusion:

120816 Colony 1.1 3.1 4.1 4.2 Colony Pcr 1-8.tif

  • 2-5 colonies 1.1, 3.1, 4.1, 4.2, 6 - 13 colony 1.1, 2.1, 2.2, 3.1, 4.1, 4.2, 5.1, 5.2

Friday, 17.08.12

Week 14 / CW 34

Monday 20.08.12

Tuesday 21.08.12

  • Ligation of BBa_K808000 upstream of BBa_K808032 in pSB1C3 ( to design BBa_K808032) from Friday, 17.08.12 worked well
  • 6 colonies are picked in order to perform a Colony PCR and for inoculation of 5 mL DYT-media-Cmp
    • colony A 4.1/2.1 ligated at 4°C
    • colony B 4.1/2.1 ligated at 37°C
    • colony C 1.1/5.1 ligated at 4°C
    • colony D 1.1/5.1 ligated at 37°c
    • colony E 4.2/2.2 ligated at 4°C
    • colony F 4.2/2.2 ligated at 37°C
  • Colony PCR
TA = 55°C, tA = 25 s, tE = 5 min
primers: VF2 & VR

Wednesday, 22.08.12

  • Miniprep of inoculated 5 mL DYT-media-Cmp with colony E,F,C,D
    • c(colony C 1.1/5.1 ligated at 4°C)=46 ng/µL
    • c(colony D 1.1/5.1 ligated at 37°c)=46 ng/µL
    • c(colony E 4.2/2.2 ligated at 4°C)=60 ng/µL
    • c(colony F 4.2/2.2 ligated at 37°C)=58 ng/µL
  • DNA Digestion of 15 µL of preped plasmids with EcoRI-HF & PstI-HF
  • Agarose gel electrophoresis with 0.8% agarose
    • ligation worked well, bands are in estimated length
    • lane 2-5: colony 4.2/2.2, lane 6-9: colony 5.1/1.1

Verdau vom Gesamtkonstrukt 4.2 2.2 und 5.1 1.1 beide temperaturen.tif

Thursday, 23.09.12

  • Miniprep of colonies from
    • c(colony C 1.1/5.1 ligated at 4°C)=147 ng/µL
    • c(colony D 1.1/5.1 ligated at 37°c)=131ng/µL
    • c(colony E 4.2/2.2 ligated at 4°C)=85 ng/µL
    • c(colony F 4.2/2.2 ligated at 37°C)=128 ng/µL
  • preparative Agarose gel electrophoresis of digested pNB-Est13 from yesterday
  • Gel extraction leads to c(pNB-Est13- cut E/P)=25 ng/µL
  • Ligation of digested pNB-Est13 in digested pSB1C3
    • 1:5 ratio is used
  • Heatshock transformation] of DH5α with 10 µL of ligation batch
  • incubation of transformed cells at 37°C over night

Friday, 24.08.12

Week 15 / CW 35

Monday, 27.08.12

Tuesday, 28.08.12

  • antibody staining
  • detection of surface expression of BBa_K808032 by using FACS
  • positive signal increases with higher temperature, staining BBa_K808000
  • protein purification of test expression without running IMAC afterwards, just using supernatant and cell debris pellet
    • 32 µL of supernatant is used
    • 3 mL pellet of each testexpression is treated with more SDS as a detergent, in order to solve membrane proteins
  • running two SDS-tris gels with an myc positive probe
  • one of these gels is used for performing a Westernblot
    • using mouse-antimyc antibody as first antibody and goat-antimouse-AP as second antibody
    • lane 2-4: supernatant of induced and disrupted cells. Level of induction decreases from lane 2 (1.5% arabinose) to lane 4 (0.5% arabinose)
    • lane 6-8: Pellet of induced and disrupted cells, treated with high SDS concentrations. Level of induction decreases from lane 6 (1.5% arabinose) to lane 8 (0.5% arabinose).
    • Lane 9-10 show myc positive probes.
    • The blot shows that our expression worked of BBa_K808032 and proofs the membrane intercalation of our chimeric protein BBa_K808030 because the blot only gives positive signals when high concentrations of detergent where used to treat the cell debris pellet.

120831 Westermblot auf Gesamt konstrukt aus DH5 alpha 5111.tif

  • both are not very good in their solution, we do it again with Laemmli gels

Wednesday, 29.08.12

  • evalutation of sequencing from last week:
    • PhoA worked
    • EstA worked
    • pNB-Est13 worked
    • RBS-PhoA-His6tag-pNBEst13-Myctag-EstA worked
    • arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA

Thursday, 30.08.12

  • running 2 SDS-Laemmli gels
    • pellet is treated with n-Dodecyl-ß-maltoside (DDM) in order to solve membrane proteins
  • one gel is used to perform a second Western blot with a myc positive probe

120831 Westermblot 2 auf Gesamt konstrukt aus DH5 alpha 5111.png

  • 2 supernatant 30°C, 3 pellet (treated with detergent) 30°C, 4 supernatant 25°C, 5 pellet (treated with detergent) 25°C, 6 supernatant 20°C, 7 pellet (treated with detergent) 20°C, 8 myc positive probe
  • as we can see, only the treated 120831 Westermblot 2 auf Gesamt konstrukt aus DH5 alpha 5111pellets show positive signals when treated with antibodies, so we have expressed our membrane protein BBa_K808032
  • now our lab can start with quantification of enzymatic activites of BBa_K808032 on PET or model substrates

Activity tests of BBa_K808032

We were able to generate our BioBricks BBa_K808030 (chimeric, membrane bound, hydrolytic protein) and BBa_K808032 ( arabinose inducible operon, regulating the expression of BBa_K808030). Funcionality of both parts will be described by a bunch of tests:


Week 1 / CW 35

Friday, 31.08.12

Component test tube 1 test tube 2 test tube 3 test tube 4
DYT-medium 8 mL 8 mL 8 mL 8 mL
PET particle yes yes yes yes
bacteria yes yes yes no
induced 1.5% L-arabinose 1.5% L-arabinose no no
  • test seemed to have worked: but an induced test tube without PET-granula was missing

Week 2 / CW 36

Tuesday, 04.09.12

Component test tube 1 test tube 2 test tube 3 test tube 4 test tube 5
DYT-medium 8 mL 8 mL 8 mL 8 mL 8 mL
PET particle yes yes no yes yes
bacteria yes yes yes no yes
induced 1.5% L-arabinose 1.5% L-arabinose 1.5% L-arabinose no no
  • test tube 3: DYT without bacteria contains Cmp, Kan, Amp
  • looks good but test tube 2 shows no significant change of colour

Wednesday, 05.09.12

Component tube 1 tube 2 tube 3 tube 4 tube 5 tube 6 tube 7 tube 8 tube 9
DYT-medium 8 mL 8 mL 8 mL 8 mL 8 mL 8 mL 8 mL 8 mL 8 mL
PET particle yes yes yes yes no no yes yes yes
bacteria yes yes yes yes yes yes yes yes no
induced 1.5% L-arabinose 1.5% L-arabinose 1.5% L-arabinose 1.5% L-arabinose 1.5% L-arabinose 1.5% L-arabinose no no no
  • test tube 9: DYT without bacteria contains Cmp, Kan, Amp
  • all induced tubes turned yellow, even without PET-granula as a substrate
  • no more avtivity tests, we are awaiting the evaluation of test expression series of the arabinose inducible promoter conjugated to GFP

Trouble shooting

Thursday, 06.09.12

Component tube 1 tube 2 tube 3 tube 4 tube 5 tube 6 tube 7 tube 8 tube 9 tube 10 tube 11
DYT-medium 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL 5 mL
PET particle yes yes yes yes yes yes no no no no no
bacteria yes yes yes yes yes yes yes yes yes yes no
induced 1% L-arabinose 1% L-arabinose 0.75% L-arabinose 0.75% L-arabinose no no 1% L-arabinose 1% L-arabinose 0.75% L-arabinose 0.75% L-arabinose no
  • test tube 11: DYT without bacteria contains Cmp, Kan, Amp
  • all induced tubes turned yellow, even without PET-granula as a substrate

Week 3 / CW 37

we stopped the trial of quantification via PET-substrates because in terms of color the change of pH value (due to acid groups that are released by degradtion) is not very exactly. In order to get reproducible data, we are using other activity assays:

Monday, 10.09.12

Tuesday, 11.09.12

120911 dh5alpha k808032 0.1 ara.jpg 120911 dh5alpha k808032 0.2 ara.jpg 120911 dh5alpha k808032 0.4 ara.jpg

Wednesday, 12.09.12

120912 Testrestriktion K808032 im ethylenglycol stamm K1 und K2 mit ecori und psti.tif

Thursday, 13.09.12

120913 dh5alpha k808032 0.1 ara.jpg 120913 dh5alpha k808032 0.2 ara.jpg 120913 dh5alpha k808032 0.4 ara.jpg

  • to perform a better expression of BBa_K808030 we will use Top10 as a host for BBa_K808032. Its unability to metabolize L-arabinose is crucial for our decision. We hope to generate good expression levels.
  • Electroporation (after washing ecletroporation cuvettes very carefully) of Top10 with BBa_K808032

Friday, 14.09.12

Saturday, 15.09.12

Week 4/ KW 38

Monday, 17.09.12

  • inoculation of 4 x 50 mL DYT-media-Cmp with Top10 stocks
  • at OD600=0,5 the cultures are incduced with:
    • 0.02% mw L-arabinose
    • 0.2% mw L-arabinose
    • 0.5% mw L-arabinose
    • one culture is not induced and will serve as a negative control
  • after 3 hours an antibody staining is performed on our expressed BBa_K808032 containing a myc-epitope
  • checking surface expression levels via flow cytometry
    • inducing worked well, we can go on with screening the enzymatic activity
E.coli Top10 carrying BBa_K808032 and induced at an OD600=0.5 with an arabinose concentration of 0.02% and no arabinose at all. The relative absorbance of 50000 cells was meassured and is shown in this plot. The induced cells show a significant difference in their absorption due to the binding of a fluorescent labeled antibody. Fot the antibody staining we used an anti-myc antibody.
E.coli Top10 carrying BBa_K808032 and induced at an OD600=0.5 with an arabinose concentration of 0.2% and no arabinose at all. The relative absorbance of 50000 cells was meassured and is shown in this plot. The induced cells show a significant difference in their absorption due to the binding of a fluorescent labeled antibody. Fot the antibody staining we used an anti-myc antibody.
E.coli Top10 carrying BBa_K808032 and induced at an OD600=0.5 with an arabinose concentration of 0.5% and no arabinose at all. The relative absorbance of 50000 cells was meassured and is shown in this plot. The induced cells show a significant difference in their absorption due to the binding of a fluorescent labeled antibody. Fot the antibody staining we used an anti-myc antibody.
E.coli Top10 carrying BBa_K808032 and induced at an OD600=0.5 with an arabinose concentration of 0.02%, 0.2%, 0.5% and no arabinose at all. The relative absorbance of 50000 cells was meassured and is shown in this plot. The induced cells show a significant difference in their absorption due to the binding of a fluorescent labeled antibody. Fot the antibody staining we used an anti-myc antibody.

Tuesday, 18.09.12

2: supernatant 0.5% arabinose, 3: treated pellet 0.5% arabinose, 4: supernatant 0.2% arabinose, 5: treated pellet 0.2% arabinose, 7: supernatant 0.02% arabinose, 8: treated pellet 0.02% arabinose, 9: supernatant no arabinose, 10: treated pellet no arabinose
















We use our induced cells (Top10) with an OD600=0.1-0.025 (resuspended in PBS-buffer) as catalysts and para-Nitrophenylbutyrate as a substrate. Induced cells have the fusion protein (BBa_K808030) expressed on their surface. The catalytical domain, formed by pNB-Esterase 13 will show hydrolytic activity we can screen for by performing a pNP-assay. The substrate will be hydrolysed, and one degradation construct is para-Nitrophenylbutyrate. If released by hydrolysis, a characeristic absorption at 405 nm can be detected and plotted against the time, resulting in straight lines. The hydrolytic activity is quantified by the slope of the respective straight line.

Plot of the bacterial pNP-Assay using induced cells (Top10) with an OD600=0.1-0.025 (in PBS-buffer) as catalysts and para-Nitrophenylbutyrate as a substrate. The hydrolytic activity is quantified by the slope of the respective straight.
Plot shows relation of the slopes from Figure 6, representing the hydrolytic activity, in coherence with the level of arabinose concentration in % used for induction.

Wednesday, 19.09.12

  • activity assays on LB Tributyrin agar worked well (incubation occured just for the night at 30°C)

120919 top10 k808032 0.02 ara.jpg 120919 top10 k808032 0.2 ara.jpg 120919 top10 k808032 0.5 ara.jpg

Tuesday, 18.09.12

120920 k808032 k808030 dh5a top10 mg1655.tif.jpg

Protein Expression

CW 20

Monday 14.05.2012

Tuesday 15.06.2012

  • one coclony of the retransformation was picked and transfered into 50 mL LB-Medium containing Cmp
  • the culture was stirred at 180 rpm at 30°C

Wednesday 16.06.12

  • centrifugation of the culture and resolving the pellet in 2 mL LB-Medium and 15% glycerine
  • seperate the cell suspension into 100 µL aliquots
  • store the stocks at -80°C

CW 24

Thursday, 14.06.2012

  • PCR for protein expression of FsC
    • each PCR is performed in 5 batches à 50 µL.
    • Parameter: TA = 57°C, tA = 35 s, tE = 65 s
  • PCR on pEST100 vector for expression with pEX vector gene of interest: FsC for designed part BBa_K808032

primers: pEX FsC His SfiI up and pEX FsC Stop SfiI lo

Friday, 15.06.2012

CW 25

Wednesday, 20.06.2012

Thursday, 21.06.2012

Friday, 22.06.2012

CW 26

Monday, 25.06.2012

Component 1:3 1:5
FsC sequence 1.12 µL 2.2 µL
pEX 0.64 µL 0.64 µL
Ligase buffer 4 µL 4 µL
T4 DNA ligase 1 µL 1 µL
H2O 33 µL 30 µL
  • Ligation time: over night

Tuesday, 26.06.2012

Wednesday, 27.06.2012

  • Two positive clones on LB Cmp plates picked for liquid culture
    50 mL DYT Cmp cultures at 180 rmp and 37°C, over night

Thursday, 28.06.2012

CW 27

Monday, 02.07.2012

Tuesday, 03.07.2012

Wednesday, 04.07.2012

Expression at 16°C/25°C
120711 FsC 16 25.jpeg
Expression at 30°C/37°C
120711 FsC 30 37.jpeg
  • The best results were maintained at an expression temperature of 30°C

Friday, 06.07.2012

CW 28

Monday, 09.07.2012

Wednesday, 11.07.2012

Thursday, 12.07.2012

120713 FsC 30.jpeg

CW 29

Monday, 16.07.2012

Tuesday, 17.07.2012

120719 Est13.jpeg

CW 31

Monday, 30.07.2012

Tuesday, 31.07.2012

120801 FsC 30.jpeg

CW 33

Friday, 17.08.2012

Saturday, 18.08.2012

120819 FsC 30.jpeg

CW 34

Wednesday, 22.08.2012

Thursday, 23.08.2012

120824 FsC 30.jpeg

Enzyme Activity Assays

To characterise our BioBricks BBa_K808026 (pNB-Est13) & BBa_K808025 (FsC) a pNP-assays are performed.

CW 37

Est13

Lineweaver-Burk plot of BBa_K808026
Km and Vmax calculation

To find Km the growth of the first seven data points were written down in a diagram. The slope of the regression line gave the speed of the hydrolysis for the single substrate concentrations. Now the value of the speed with the associated concentration of substrate were plotted in a Lineweaver-Burk diagram. At the point where the regression line is crossing the x-axis the x-coordinate amounts to -1/Km. So Km is about 400µM. To calculate the value of Vmax we used the point where the plotted line crosses the y-axis. The associated y-coordinate is 1/Vmax. Consequently Vmax for 50nM Est13 is 0,002798 1/s.

Kcat calculation

The value Kcat is calculated through dividing Vmax by the enzyme concentration of 50nM. Kcat counts 0,055951 1/mol*s.

FsC


Lineweaver-Burk plot of BBa_K808025
Km and Vmax calculation

To find Km the growth of the first twelve data points were written down in another diagram. The slope of the regression line gave the speed of the hydrolysis for the single substrate concentrations. Now the value of the speed with the associated concentration of substrate were plotted in a Lineweaver-Burk diagram. At the point where the regression line is crossing the x-axis the x-coordinate amounts to -1/Km. So Km is about 400µM. To calculate the value of Vmax we used the point where the plotted line crosses the y-axis. The associated y-coordinate is 1/Vmax. Consequently Vmax for 5nM FsC is 0,001467 1/s.

Kcat calculation

The Kcat value is calculated through dividing Vmax by the enzyme concentration of 5nM. Kcat counts 0,293395 1/mol*s.

FsC with ehtylenglycol

Our Simulation lab was able to show , theoratically, a relation between an decrease of enzyme activity and an increase of ethylenglycol concentration. This could be a hint for the mysterious loss of hydrolysis when the degradation rate reaches 5% of PET foil weight as it is described in the literature (Ronkvist, Å. M., Xie, W., Lu, W., & Gross, R. a. (2009). Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate). Macromolecules, 42(14), 5128–5138. doi:10.1021/ma9005318). We tried to proof their theory of potentially higher ethylenglycol concentration surrounding the PET foil. An increasing amount of ehtylenglycol should hamper the hydrolytic activity of FsC in a pNP-assay.

Procedure

The lab test procedure was nearly equal to the normal kinetics records with only one difference. The reaction solution consisted of different composites of PBS buffer and ethylenglycol (0 - 20%). All records were done at a concentration of 1mM of 4-Nitrophenyl butyrate in the experiment.

Records of the different ethylenglycol concentrations and the associated speeds of hydrolisis in a diagram
Plot of enzyme activity related to an increasing ehtylenglcol concentration
Interpretation

As you can see there is a stagnation of the speed of hydrolysis of the ester caused by an increasing concentration of ethylenglycol. But by that you cannot tell that ethylenglycol inhibits the FsC, because the hydrolysis of the ester rose with a higher concentration of ethylenglycol in the negative samples. So it could be that the ethylenglycol starts a concurrent reaction or blocks the 4-Nitrophenyl butyrate.

Plot of "activty" in the negative sample: pNPB & ethylenglycol

CW 36 - 38

PET degradation

Principle and conditions
principle of hyrolytic PET degradation. Conditions: Room temperature (~ 22°C) pH = 7.4

Procedure

Round about 1g of PET granulate is added in each two falcon tubes to between 15 and 20 mL water of pH = 7.4 and several drops of bromothymol blue. In one of these falcon tubes Est13 stock solution is added until a concentration of 2,5 µM is reached. For 15 days the solutions are incubated at room temperature. At nine specific days of these the enzymatic solutions and the negative sample are photographed, to control the shift of pH.

Results

In all photos the degradation with Est13 is in the left falcon tube.

Day1:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control
Day2:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control
Day3:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control
Day7:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control
Day9:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control
Day10:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control
Day13:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control
Day15:Degradation of PET. Left: With Est13(2,5µM) Right: Negative control



The photos show a decrease of the pH-value over time in the sample containing Est13. The one without this enzyme remains unchanged. So it is concluded that Est13 degrades PET granulate.

CW34 - CW35

Degradation of monomethyl terephthalate over time

Principle and conditions

Principle of degradation of monomethyl terephthalate.png
Conditions: Room temperature (~22°C)

Procedure

Three different samples of 30 mL dest. water at a pH of 7.4 are incubated for 7 days with a concentration 500µM of monomethyl terephthalate (solved in methanol) and following ingredients:

1. 30µL of FsC (concentration in the incubated solution: ~0,8µM)
2. 30µL of Est13 (concentration in the incubated solution: ~0,8µM)
3. Nothing

To control the shift of the pH during the estercleavage some drops of bromothymol blue are added.

Results

Following pictures show the shift of the pH over 7 days (The degaradation with Est13 is in the left falcon tube and with FSC in the middle one):

Day1: Degradation of monomethyl terephthalate. Left: with Est13(~0,8µM) Middle: with FSC(~8µM) Right: Negativ sample
Day8: Degradation of monomethyl terephthalate. Left: with Est13(~0,8µM) Middle: with FSC(~8µM) Right: Negativ sample

So you can see that both enzymes catalyzed the cleavage of the ester in a few days. In 7 days the pH-value shifted about one unit.

CW38

Degradation of 4-nitrophenyl-3-phenylpropanoate and bis(4-nitrophenyl) succinate

Principles, conditions and method
Principle of degradation of 4-nitrophenyl-3-phenylpropanoate
Principle of degradation of bis(4-nitrophenyl) succinat

Conditions and method are listed by the pNP-assay.

Procedure

The procedure is the same like in the pNP-assay. Only if bis(4-nitrophenyl) succinate is added, add 900µL of reagent A to 100µL of reagent B in step 1.

Results
Plot of enzyme activities on 4-nitrophenyl-3-phenylpropanoate and bis(4-nitrophenyl) succinate
Interpretation

The result shows that Est13 can catalyze the cleavage of both esters. FsC cannot do this in the given conditions. It is not clear what the reasons for the deactivation of FsC are. Maybe DMSO and/or methanol inhibit the enzyme or it is even not able to catalyze the hydrolysis.

Retrieved from "http://2012.igem.org/Team:TU_Darmstadt/Labjournal/Degradation"