Notebook

LABORATORY NOTEBOOK

GENERAL NOTEBOOK

This notebook presents fourteen weeks of our laboratory work during which Marta, Adam, Maciej, Mikołaj and Julia developed the idea of enzymatic decomposition of harmful phthalates. You can also find information about our other activities. For more information about experiments and protocols, please visit the Experiments tab.

JUNE

In the first week, we prepared fresh competent cells, LB medium, LB-Agar, and our laboratory space to start the transformation. Basic parts were designed with respective prefixes and suffixes and tested ligation in silico.

In the lab: Marta, Mikołaj

27.06.2023
  • E. coli DH5α DH5αF’ cells from the freezer (– 80 °C) were plated on TSB medium - streak plate method.
28.06.2023
  • Preparation of LB medium and LB-Agar.
  • Passage of E. coli DH5α from the plate to liquid medium.
29.06.2023
  • Competent cells were prepared using the E. coli Transformer Express Kit according to the manufacturer's protocol [protocol 1].
  • Preparation of glycerol bacteria stock of E. coli DH5α DH5αF’ cells.

JULY

First transformation with usage iGEM distribution kit, this week We succeed in transformation of two parts, and we performed transformation with different strain of competent cells. Sequences of designed basic parts were optimized and ordered.

In the lab: Marta, Mikołaj, Mateusz

4.07.2023
  • Preparation of ampicillin stocks 100 mg/ml.
  • Preparation of LB-Agar plates with ampicillin (final concentration 100 mg/ml).
  • First transformation with usage E. coli Transformer Express Kit [protocol 2]. We chose the following parts from list (Table 1): promoter, vector pUC, RBS, GFP, linkerDF, AmilCP.
5.07.2023
  • First success - we managed to transform the vector pUC construct.
  • Bacteria transformed with the vector pUC element were plate into LB liquid medium with ampicillin (100 μg/ml).
6.07.2023
  • Preparation of glycerol bacteria stock of E. coli DH5α harboring vector pUC.
  • Transformation of E. coli DH5α with plasmid from iGEM (Table 1): promoter, vector pUC, RBS, GFP, linkerDF, AmilCP [protocol 2].
  • Ordering sequences of 9 basic parts from IDT in form of gBlocks.
7.07.2023
  • Transformation result: we managed to transform GFP construct, a single colony was plated into new LB-Agar plates with kanamycin (30 ug/ml) and ampicillin (100ug/ml).
  • Preparation of ampicillin stocks (100 mg/ml) and kanamycin (30 mg/ml).
  • Transformation of NEB10- beta competent cell [protocol 3], we chose the following parts from list (Table 1): promoter, RBS, linkerDF, AmilCP.
We had to consider the possible reason of low efficiency of transformation and find a solution to improve our result. We decided to try electroporation transformation.

In the lab: Marta and Adam

10.07.2023
  • Results from transformation- we did not observe colonies.
  • LB-agar preparation.
11.07.2023
  • Plasmids isolation [protocol 4] - vector pUC and GFP.
  • Preparation of glycerol bacteria stock of E. coli DH5α harboring GFP.
  • We performed agarose gel electrophoresis to confirm efficiency and purity of plasmid isolation.
  • Electroporation transformation of E. coli DH5α with promoter and cleaning the cuvettes after electroporation transformation [protocol 5].
13.07.2023
  • Result from electroporation transformation of E. coli DH5α with promoter: we observed 4 colonies which we plated on LB with higher ampicillin concentration (100 mg/ml).
  • We performed agarose gel electrophoresis to confirm efficiency and purity of plasmid isolation - vector pUC and GFP.
14.07.2023
  • We performed agarose gel electrophoresis to confirm efficiency and purity of plasmid isolation - vector pUC and GFP – Rerun with usage higher marker volume.
  • E. coli DH5α harboring vector pUC were plated on LB-Agar plates with ampicillin (100 mg/ml).
Finally, we found a solution to improve our result. In previous transformations we used too little DNA, centrifuging the plate before collection turned out to be the solution to our problem.

In the lab: Maciej, Adam and Julia

17.07.2023
  • Inoculation of E. coli DH5α harboring vector pUC into liquid media with ampicillin 100 mg/ml.
  • 5x TBE pH 8,3 Buffer preparation (Trizma base; 2,75 % Boric acid; 0,5 M EDTA pH 8,0).
  • Preparation of plate with ampicillin (100 mg/ml).
18.07.2023
  • Preparation of glycerol bacteria stock of E. coli DH5α harboring promoter.
  • Plasmids isolation [protocol 4] - promoter.
  • We performed agarose gel electrophoresis to confirm efficiency and purity of plasmid (promoter) isolation.
  • Transformation of NEB10-beta competent cell [protocol 3] we chose the following parts from list (Table 1): linkerEF, RBS, linkerDF, pUC19.
19.07.2023
  • E. coli DH5α harboring plasmid: linkerEF, RBS, linkerDF, pUC19 (from transformation 18.07) were plated on LB-Agar plates with ampicillin (100 mg/ml) - streak plate method.
  • Restriction analysis of promoter with BsaI HF enzymes [protocol 6].
20.07.2023
  • Transformation of NEB10-beta competent cell [protocol 3], we chose the following parts from list: linkerDT4, linkerDT4tev, linkerT4T5, linkerT5Ehis and pUC19 as control.
  • E. coli NEB10-beta harboring plasmid: linkerEF, RBS, linkerDF, pUC19 were inoculated into liquid media with ampicillin (100 mg/ml).
21.07.2023
  • Plasmids isolation [protocol 4] - linkerEF, RBS, linkerDF.
  • E. coli NEB10-beta harboring plasmid: linkerDT4, linkerDT4tev, linkerT4T5, linkerT5Ehis, pUC19 were plated into liquid media with ampicillin (100 mg/ml).
  • Preparation of glycerol bacteria stock of E. coli NEB10-beta linkerEF, RBS, linkerDF.
We were keeping transforming the subsequent components required to ligate our designed construct. Sequences of designed devices were optimized and ordered.

In the lab: Julia, Adam and Mikołaj

24.07.2023
  • E. coli NEB10-beta harboring plasmid: linkerDT4, linkerT5Ehis, linkerDT4, linkerDT4tev were inoculated into liquid media with ampicillin (100 mg/ml).
  • Determination of DNA concentration with a use of spectrophotometric analysis – nanodrop [Protocol 7].
  • Restriction analysis of plasmids: promoter, RBS, linkerDF, linkerEF [Protocol 6]. Result was checked by performing agarose gel electrophoresis.
  • Ordering sequences of 3 devices from IDT in form of gBlocks Gene Fragments.
25.07.2023
  • Plasmids isolation [protocol 4] linkerDT4tev and linkerDT4.
  • Preparation of glycerol bacteria stock of E. coli NEB10-beta harboring DT4_32, linkerDT4, linkerT4T5, linkerT5Ehis.
26.07.2023
  • LB-Agar plates with kanamycin 30 mg/ml preparation.
  • Trial ligation of construct with GFP with using following parts: promoter, RBS, linkerDF, GFP, vector pUC 19 [protocol 8].
  • Transformation of E. coli DH5α [protocol 2] with plasmid after ligation.
  • Transformation of E. coli BL21 (DE3) competent cell [protocol 9] with plasmid after ligation.
  • E. coli NEB10-beta harboring plasmid: linkerT4T5 and linkerT5Ehis were inoculated into liquid LB media with ampicillin (100 ug/ml) for plasmid isolation.
27.07.2023
  • We succeed in transformation of both strains E. coli DH5α and E. coli BL21 (DE3).
  • LB-Agar plates media with kanamycin 30 mg/ml preparation.
  • 1 M IPTG stock preparation.
  • Inoculation of E. coli DH5α harboring GFP construct into liquid media with kanamycin 30 mg/ml.
  • E. coli DH5α and E. coli BL21 (DE3) harboring plasmid with GPF after transformation were plated into LB-Agar plates with kanamycin 30 mg/ml and IPTG 1 mM and as negative control we used LB-Agar plates with ampicillin100 mg/ml – after the ligation of GFP construct the gene encoding ampicillin resistance should be cut out and we should not observe colonies on plate with ampicillin.
  • Inoculation of E. coli BL21 (DE3) harboring GFP construct into liquid media with kanamycin (30 mg/ml) to trial overproduction.
  • Plasmids isolation [protocol 4] linkerT4T5 and linkerT5Ehis.
  • Restriction analysis of plasmids: linkerDT4, DT4_357, linkerT4T5, linkerT5Ehis [Protocol 6] The results was checked by performing agarose gel electrophoresis.

AUGUST

This week Adam, Maciej and Julia started with ligation of our designed construct, we also tried to do overproduction of trial construct with GFP.

In the lab: Adam, Maciej, Julia.

31.07.2023
  • Determination of DNA concentration (linkerDT4, linkerDT4tev, linkerT4T5, linkerT5Ehis) with a use of spectrophotometric analysis – nanodrop [protocol 7].
  • Suspending gBlocks in water [protocol 10].
  • Preparation for ligation – calculations.
  • We transfer around 20 colonies of E. coli DH5α and E. coli BL21 (DE3) harboring plasmid with GPF after transformation which were plated into LB-Agar plates with kanamycin (30 mg/ml) and LB-Agar plates with ampicillin (100 mg/ml) to confirm lack of ampicillin resistance after ligation.
  • Ligation of our designed construct [protocol 8]
01.08.2023
  • Transformation of E. coli NEB10 beta [protocol 3] with plasmid after ligation.
  • LB-Agar plates with kanamycin (30 mg/ml) preparation.
  • Single colony of E. coli harboring the plasmid with GFP was inoculated into liquid media with kanamycin (30 mg/ml).
02.08.2023
  • Transformation of E. coli NEB10 beta with plasmid after ligation result – we managed to transform all construct, which we had designed.
  • Colonies from transformation were transferred into LB-Agar plates with kanamycin (30 μg/ml) and LB-Agar plates with ampicillin (100 μg/ml) to confirm lack of ampicillin resistance after ligation. This step allows us to confirm successful ligation.
  • The overproduction of GFP in E. coli BL21 (DE3) harboring vector pUC-GFP trial ended in failure [protocol 11].
03.08.2023
  • Colonies from transformation which were transferred into LB-Agar plates with kanamycin 30 mg/ml and LB-Agar plates with ampicillin 100 mg/ml did not grow on ampicillin media. This step indicated successful ligation.
  • A single colony E. coli NEB10 harboring plasmid encoding our ligation product (Dcx1-DocXylY, Dcx2-DocXylY, Dcx3-DocXylY, Dcx4-DocXylY, PnbA-DocScaB, ScfL1) was resuspend in liquid LB with kanamycin 30 mg/ml and was incubated in 37 °C overnight.
04.08.2023
  • Plasmids isolation [protocol 4] Dcx1-DocXylY, Dcx2-DocXylY, Dcx3-DocXylY, Dcx4-DocXylY, PnbA-DocScaB, ScfL1.
  • Preparation of glycerol bacteria stock of E. coli NEB10 harboring Dcx1-DocXylY, Dcx2-DocXylY, Dcx3-DocXylY, Dcx4-DocXylY, PnbA-DocScaB, ScfL1
  • Restriction analysis of plasmids: Dcx1-DocXylY, Dcx2-DocXylY, Dcx3-DocXylY, Dcx4-DocXylY, PnbA-DocScaB, ScfL1 [Protocol 6]. We made mistake, we used Bsal instead of SapI.
  • A single colony E. coli NEB10 harboring plasmid encoding our vector pUC was resuspend in liquid LB with ampicillin 100 mg/ml and was incubated in 37 °C overnight.
We made a decision to try different vector than vector pUC for ligation, it’s turned on that at our faculty are some possible plasmids which we can use. Additionally, we had the opportunity to use vector, which is better for protein expression. Simultaneously, we decided to keep attempting to use vector pUC. Our designed constructs were transferred into pJUMP vector, which is available on iGEM distribution kit.

In the lab: Maciej, Adam and Julia

07.08.2023
  • Chloramphenicol stock preparation.
  • Plates with kanamycin 30 mg/ml, Chloramphenicol 100 mg/ml, Ampicillin 100 mg/ml preparation.
  • Restriction analysis of plasmids: Dcx1-DocXylY, Dcx2-DocXylY, Dcx3-DocXylY, Dcx4-DocXylL, PnbA-DocScaB, ScfL1 [Protocol 6]. This time with proper enzyme- Sapl.
  • Isolation of plasmids from overnight six cultures with vector pUC and restriction analysis using BsaI.
08.08.2023
  • Plasmids isolation from E. coli NEB10 harboring plasmid encoding vector pUC [protocol 4].
  • Restriction analysis of plasmids from E. coli NEB10 harboring plasmid encoding vector pUC. Since last time our restriction analysis was inconclusive, we decided to isolate vector pUC plasmid and perform analysis one more time.
  • After consolation with dr Katarzyna Węgrzyn we planned to try different vector than vector pUC, it turned on that on our faculty are some possible plasmids which we can use. Additionally, we had the opportunity to use vector, which is better for protein expression. Simultaneously, we decided to keep attempting to use vector pUC.
  • We found available plasmid (pET, pBAD24, pBAD30, pCOLADuet) and enzymes which cut leaving blunt ends.
09.08.2023
  • We chose pBAD24. However, we also had a plan to isolate any potential plasmids.
  • We were working on new blunt ends ligation protocol [protocol 12].
10.08.2023
  • Our constructs which we ordered from IDT finally arrived - Dcx1-DocXylY, PnbA-DocScaB, EstJ6-DocScaB inserted vector pUC.
  • Suspension of IDT constructs in water [protocol 10].
  • Ligation of our constructs which we ordered from IDT [protocol 8 (for library preparation)].
  • Following ligation, transformation of E. coli BL21 (DE3) with the target construct.
  • Plasmid isolation [protocol 4] - pBAD24, pBAD30, pCOLADuet-1, pET15b.
  • Determination of DNA concentration (pBAD24, pBAD30, pCOLADuet-1, pET15b) with a use of spectrophotometric analysis – nanodrop [protocol 7].
  • Blunt ends ligation with EcoRV and shrimp phosphatase and T4 ligase [protocol 12].
11.08.2023
  • Colonies from transformation (10.08) were transferred into LB-Agar plates with kanamycin (30 mg/ml) and LB-Agar plates with ampicillin (100 mg/ml) to verify successful ligation.
  • We also decided to transfer our designed construct into pJUMP vector (pJUMP 23-1A, pJUMP 26-1A, pJUMP 28-1A, pJUMP 29-1A -Table 1) for this reason we did four ligations of pJUMP from iGEM distribution kit [protocol 9]. Following ligation, transformation of E. coli DH5α with the target constructs.
  • Transformation of E. coli Rosetta with ligates of “Blunt Ends” inserts and pBAD24 from the previous day.
The GFP expression in E. coli DH5alpha with pJUMP ended with success. That result indicated that working with pJUMP vector is good choice and isolated different pJUMP vector.

In the lab: Maciej and Adam

14.08.2023
  • LB-Agar plates with ampicillin 100 mg/ml preparation.
  • Repetition of transformation of E. coli Rosetta with ligates of “Blunt Ends” inserts and pBAD24, from previous transformation we obtain just two colonies. These two colonies were transfer into LB-Agar plates with ampicillin (100 mg/ml) to check efficiency of ligation.
  • The transformation from 11/08 of the pJUMP plasmids to DH5alfa was successful.
15.08.2023
  • Inoculation of an overnight culture of E. coli DH5α with pJUMP plasmids after transformation from 11/08.
  • Inoculation of the night culture of E. coli Rosetta with the blunt-ended ligation mixture from 11/08 and 14/08
  • Screening of colonies obtained during transformation of E. coli BL21 bacteria with the vector pUC + ligation mixture of constructs (Dcx1-DocXylY, PnbA-DocScaB) showed that the vast majority of colonies grow on kanamycin, but do not grow on ampicillin, which indicates a positive ligation result:
16.08.2023
  • We compared the amount of GFP produced during overnight culture for different pJUMP plasmids and we chose the most promising vector - pJUMP 23-1A.
  • Plasmids isolation from E. coli DH5α harboring plasmid encoding vector pUC with construct (Dcx1-DocXylY, PnbA-DocScaB, EstJ6-DocScaB), pBAD24 with construct (+Dcx1-DocXylY, PnbA-DocScaB) and pJUMP (pJUMP 23-1A, pJUMP 26-1A, pJUMP 28-1A, pJUMP 29-1A) [protocol 4].
  • Determination of DNA concentration (of isolated plasmids) with a use of spectrophotometric analysis – nanodrop [protocol 7].
17.08.2023
  • Restriction analysis of pBAD24 (with inserted constructs +Dcx1-DocXylY + PnbA-DocScaB) with EcoRI and EcoRII.
  • The result of the restriction analysis of vector pUC with construct (+Dcx1-DocXylY, + PnbA-DocScaB, + EstJ6-DocScaB) allowed us to confirm the success of ligation. Unfortunately, the result of the restriction analysis of pBAD24 turned out to be inconsistent with expectations.
  • Trial overproduction of inserts (vector pUC and pBAD24) in E. coli BL21.
  • Transformation of E. coli BL21 with the mixture after ligation of the pJUMP 23-1a plasmid with the Dcx1-DocXylY, PnbA-DocScaB and EstJ6-DocScaB constructs.
  • Preparation of buffer to perform SDS-page electrophoresis.
  • Ligation of PnbA-DocScaB, EstJ6-DocScaB and Dcx1-DocXylY into pJUMP23-1A [protocol 8].
18.08.2023
  • Preparation of polyacrylamide gels for electrophoresis.
  • The overproduction of Dcx1-DocXylY, PnbA-DocScaB, and EstJ6-DocScaB inserts in vector pUC was analyzed using SDS PAGE, due to mistake in sample preparation the result was inconclusive.
  • Screening of BL21 transformants potentially containing pJUMP 23-1A +Dcx1-DocXylY, PnbA-DocScaB, EstJ6-DocScaB on a kanamycin plate.
Our main goal for this week was to do successful overproduction of enzymes, which plays pivotal role for our project concept. We also started to analyze samples with HPLC. Maciej and Adam had the great opportunity to take part in nanopore sequencing workshop.

In the lab: Adam and Maciej

21.08.2023
  • Inoculation of overnight culture with constructs in pJUMP23
  • A single colony E. coli BL21 harboring plasmid encoding pJUMP 23-1A +Dcx1-DocXylY, PnbA-DocScaB, EstJ6-DocScaB was resuspend in liquid LB with kanamycin 30 mg/ml and was incubated in 37 °C overnight.
  • A single colony E. coli BL21 harboring plasmid encoding vector pUC +Dcx1-DocXylY, PnbA-DocScaB, EstJ6-DocScaB was resuspend in liquid LB with kanamycin (30 mg/ml) and was incubated in 37 °C overnight.
  • Confirmation of the absence of ampicillin resistance of ScfL1 after ligation.
22.08.2023
  • Preparation of Laemmli Buffer [protocol 13].
  • Ligation of ScfL1f [protocol 8].
  • LB-Agar plates with kanamycin 30 mg/ml preparation.
  • Plasmids isolation from E. coli DH5α harboring plasmid encoding pJUMP (23-1A, 26-1A, 28-1A, 29-1A Table 1) [protocol 4].
  • Determination of DNA concentration (of isolated plasmids) with a use of spectrophotometric analysis – nanodrop [protocol 7].
  • Restriction analysis of pJUMP23-1A (Dcx1-DocXylY, PnbA-DocScaB i EstJ6-DocScaB) with XbaI and BsaI as a negative control [protocol 6].
  • A single colony E. coli BL21 harboring plasmid encoding pJUMP231-A + Dcx1-DocXylY/ PnbA-DocScaB /EstJ6-DocScaB was resuspend in liquid LB with kanamycin 30 mg/ml and was incubated in 37 °C overnight.
  • Preparation of glycerol bacteria stock of E. coli BL21 harboring pJUMP23-1A + Dcx1-DocXylY/ PnbA-DocScaB /EstJ6-DocScaB
  • Second overproduction of inserts (+ Dcx1-DocXylY/ PnbA-DocScaB /EstJ6-DocScaB) vector pUC in E. coli BL21 (DE3). [protocol 14].
23.08.2023
  • Consultation about working with HPLC with dr Marcel Thiel
  • The overproduction of Dcx1-DocXylY, PnbA-DocScaB, and EstJ6-DocScaB inserts of vector pUC in E. coli BL21 (DE3) was analyzed using SDS PAGE. For Dcx1-DocXylYand PnbA-DocScaB we can confirm overproduction.
  • Transformation of E. coli BL21 (DE3) with the ligation mixture after the second ScfL1 ligation attempt.
  • DBP solubility has been checked in PBS for HPLC samples.
24.08.2023
  • DBP analysis on HPLC for standard curve preparation.
  • Restriction analysis of pJUMP23-1A (Dcx1-DocXylY, PnbA-DocScaB and EstJ6-DocScaB) with XbaI and BamHI [protocol 6].
  • The overproduction of Dcx1-DocXylY, PnbA-DocScaB, and EstJ6-DocScaB (pJUMP in E. coli BL21 (DE3)) was analyzed using SDS PAGE [protocol 14].
  • The production was successful.
  • Isolation of 6 plasmids from cells transformed with first ScfL1 after ligation [protocol 4].
  • Preparation of glycerol bacteria stock of E. coli NEB10-beta 10 harboring ScfL1 after ligation.
  • Colonies from transformation (23.08) were transferred into LB-Agar plates with kanamycin (30 mg/ml).
25.08.2023
  • Nanopore sequencing workshop.
We were trying to optimize overproduction of enzymes in E. coli. Simultaneously, we was keeping testing samples on HPLC.

In the lab: Adam and Maciej

28.08.2023
  • A single colony E. coli BL21 harboring plasmid- product of second ligation of ScfL1foldin was resuspend in liquid LB with kanamycin (30 mg/ml) and was incubated in 37 °C overnight.
  • A single colony E. coli BL21 harboring plasmid encoding pJUMP231 + Dcx1-DocXylY/ PnbA-DocScaB /EstJ6-DocScaB was resuspend in liquid LB with kanamycin (30 μg/ml) and was incubated in 37 °C overnight.
29.08.2023
  • Plasmids isolation from E. coli BL21 harboring plasmid encoding pJUMP-1A after second ligation of ScfL1foldin [protocol 4].
  • Determination of DNA concentration (of isolated plasmids) with a use of spectrophotometric analysis – nanodrop [protocol 7].
  • Preparation of glycerol bacteria stock of E. coli BL21 (DE3) harboring pJUMP23-1A with ScfL1f after ligation.
  • A single colony E. coli BL21 harboring plasmid - product of second ligation of ScfL1 into pJUMP23-1A was resuspend in liquid LB with kanamycin (30 mg/ml) and was incubated in 37 °C overnight.
  • A single colony E. coli BL21 harboring plasmid- Dcx1-DocXylYand PnbA-DocScaB w pJUMP23-1A was resuspend in liquid LB with kanamycin (30 mg/ml) and was incubated in 37 °C overnight.
  • Restriction analysis pJUMP23-1A+S with BsaI and SapI enzymes.
  • We performed agarose gel electrophoresis to confirm validity of our construct’s insertion into pJUMP23.
  • Consultations with prof. Ołdziej about the solubility standards.
  • Preparation of stocks of HPLC standards.
  • MBP, BB, BA and PA solutions were prepared at a concentration of 1 mM, each in 3 versions: 100 % PBS, 20 % ACN/PBS, 100 % ACN.
30.08.2023
  • Rerun of agarose gel electrophoresis was performed to confirm validity of our construct’s insertion into pJUMP23.
  • HPLC sample preparation.
  • Preparation of polyacrylamide gels for electrophoresis [protocol 13].
  • The overproduction of Dcx1-DocXylY, PnbA-DocScaB, and EstJ6-DocScaB inserts of pJUMP23 in E. coli BL21 (DE3) was analyzed using SDS PAGE [protocol 14].
  • Unfortunately, we did not observe Dcx1-DocXylY, PnbA-DocScaB or EstJ6-DocScaB overproduction.
  • A single colony E. coli BL21 harboring plasmid- Dcx1-DocXylY and PnbA-DocScaB w pJUMP23-1A was resuspend in liquid LB with kanamycin 30 mg/ml and was incubated in 37 °C overnight.
  • Preparation of trial overproduction of pJUMP23+Dcx1-DocXylY, PnbA-DocScaB and EstJ6-DocScaB with the addition of CaCl2.
  • ScfL1 ligation test no. 3 – preparations optimalisation
  • Analysis of samples, which were run on HPLC.
01.09.2023
  • Plasmids isolation from E. coli BL21 harboring plasmid encoding promoter [protocol 4]. -for ScfL1 ligation
  • Determination of DNA concentration (of isolated plasmids) with a use of spectrophotometric analysis – nanodrop [protocol 7].
  • Overproduction of Dcx1-DocXylY, PnbA-DocScaB and EstJ6-DocScaB (trial 2, this time we changed incubation time to 3 hours at 37 °C) – lower yield overproduction than previous which was incubated overnight, and there is no visible overproduction of EstJ6-DocScaB.
  • Third ligation trial with use of following parts: Promoter, RBS, ScfL1, linkerDT4, linkerT4T5, linkerT5Ehis, linkerEF, pJUMP 23-1A (Table 1)
  • Checking whether 1mM DBP precipitates from the solution.
  • Preparation of polyacrylamide gels for electrophoresis.
  • A single colony E. coli BL21 harboring plasmid- Dcx1-DocXylY and PnbA-DocScaB w pJUMP23-1A was resuspend in liquid LB with kanamycin (30 mg/ml) and was incubated in 37 °C overnight.

SEPTEMBER

Successful overproduction and purification of enzymes crucial for prove of our concept. Primers needed for PCR were designed and sequence of designed device were designed and ordered.

In the lab: Marta, Mikołaj, Adam and Maciej

02.09.2023
  • Dcx1-DocXylYPnbA-DocScaB overproduction in E. coli BL21 (DE3) – we decided to focus on these enzymes.
03.09.2023
  • Overproduction results: we harvest 13 g of Dcx1-DocXylY and 15 g of PnbA-DocScaB and was frozen in liquid nitrogen.
  • The overproduction of Dcx1-DocXylY, PnbA-DocScaB, and inserts in pJUMP23 was analyzed using SDS PAGE.
  • Transformation of E. coli NEB10-beta bacteria with mixtures after 3rd ScfL1 ligation [protocol 3].
04.09.2023
  • Collection of data from previous HPLC analysis.
  • Preparation of 10 mM stock standards in ACN.
  • Preparation of samples for screening of standard concentrations. 10 different concentrations of the standard mixture were prepared in a volume of 1 ml to check the approximate linearity ranges of their concentrations and detection thresholds.
  • Screening of colonies after transformation after 3rd ScfL1 ligation.
  • Preparations for protein purification [protocol 14].
  • Preparation of imidazole stock 1 M pH 7,4.
  • PnbA-DocScaB purification: bacterial lysis by sonication, centrifugation, supernatant was collected and frozen in liquid nitrogen (the same step for Dcx1-DocXylY purification).
  • The presence of protein in supernatant was checked by SDS PAGE.
05.09.2023
  • Ordering sequence of 1 device from TWIST Bioscience in form of Gene Fragments.
06.09.2023
  • Sample preparation for the DBP standard curve.
  • Overproduction of ScfL1 in screening tubes.
07.09.2023
  • The overproduction of ScfL1 after third ligation was analyzed using SDS PAGE [protocol] 14.
  • PnbA-DocScaB and Dcx1-DocXylY were purified by Immobilized Metal Affinity Chromatography (His60 Ni Superflow Resin TaKaRa).
  • Combined fraction was diluted to final BF overnight.
08.09.2023
  • Part of purified protein was frozen in liquid nitrogen and the rest was incubated with TEV protease.
  • After TEV cleavage the part with Histag was separated by NiNTA column.
  • Ordering primers from IDT.
The SDS PAGE was performed to confirm successful protein purification. We also manage to carry out enzyme activity tests.

In the lab: Adam

11.09.2023
  • Preparation of a standard curve for HPLC.
  • Preparation of polyacrylamide gels for electrophoresis.
12.09.2023
  • Running a third repeat of the standard curve on the HPLC.
  • A single colony E. coli BL21 harboring pJUMP 23-1A - ScfL1 was resuspend in liquid LB with kanamycin (30 mg/ml) and was incubated in 37 °C overnight.
  • SDS page for all protein purification steps was performed to check efficiency of the purification [Protocol 13].
13.09.2023
  • PnbA-DocScaB enzyme test- we run sample after enzyme reaction on HPLC.
  • Plasmids isolation from E. coli BL21 harboring pJUMP after ScfL1 ligation [protocol 4].
  • Preparation of LB medium and LB-Agar.
  • To confirm TEV cleavage of PnbA-DocScaB SDS PAGE was perform.
14.09.2023
  • Preparing primers to check whether the ScfL1 ligation process was carried out correctly.
  • We perform enzyme activity tests - PnbA-DocScaB enzyme test for BB and PnbA-DocScaB enzyme assay for DBP.
  • Checking and analyzing results from HPLC – enzyme test activity.
  • Determination of DNA concentration with a use of spectrophotometric analysis – nanodrop [Protocol 7].
  • Agarose electrophoresis of after plasmids isolation from cells after the second ligation of ScfL1.
  • Transferring the column to different HPLC (without PDA).
  • Running the samples from enzymatic tests of PnbA-DocScaB with DBP and BB.
Marta and Adam evaluated the effectiveness of the ScfL1 ligation and transformation. Test of PnbA-DocScaB enzymatic activity was performed to confirm our previous results.

In the lab: Marta, Julia Adam and Maciej

18.09.2023
  • Running 400 mM mix on new HPLC to compare results between HPLCs.
  • A single colony E. coli BL21 harboring plasmid Dcx1-DocXylY- pJUMP 23-1A was resuspend in liquid LB with kanamycin 30 mg/ml and was incubated in 37 °C overnight.
  • Colony PCR – checking the presence of ScfL1 in transformed bacteria.
19.09.23
  • Dcx1-DocXylY overproduction in E. coli BL21 [protocol 14]
  • Agarose gel electrophoresis of 2% of samples from 18.09, we can observe product.
20.09.2023
  • Colony PCR for 5 colonies harboring the pJUMP 23-1A vector with ScfL1.
  • PCR of pJUMP23-A plasmids containing PnbA-DocScaB
21.09.2023
  • Agarose gel electrophoresis – samples from 20.09.
  • The result coincides with the predictions, so it can be assumed that each of the tested colonies carries a plasmid containing the ScfL1 sequence.
22.09.2023
  • Test of PnbA-DocScaB enzymatic activity – repetition 2 and 3.
functional name our abbreviation Short name (unified) by iGEM ID
promotor
 Prom_296 AB_T7_lacO BBa_J435350
vector pUC VecT_295 AF_high copy
(pUC)
ori/KanR		 BBa_J435330
RBS RBS_348 BC_BCD23 BBa_J435385
linkerDT4 DT4_274 DT4_GS
flexible linker		 BBa_J435309
linkerDT4tev DT4_357 DT4_TEV BBa_J435395
linkerEF Term_324 EF_T7term BBa_J435361
linkerT4T5 T4T5_312 T4T5_GS
flexible		 BBa_J435349
linkerT5Ehis T5E_332 T5E_6H BBa_J435369
pJUMP 23-1A 23-1A pJUMP23-1A(sfGFP) BBa_J428347
pJUMP 26-1A 26-1A pJUMP26-1A(sfGFP) BBa_J428350
pJUMP 28-1A 28-1A pJUMP28-1A(sfGFP) BBa_J428353
pJUMP 29-1A 29-1A pJUMP29-1A(sfGFP) BBa_J428341
GFP Gfp_293 CD_fuGFP BBa_J435328
linkerDF DF_355 DF_TEVcut_H
is_Stop_TZ		 BBa_J435393
AmilCP Amil_122 CD_AmilCP BBa_J435338
28.11.2022 - 12.12.2022
  • Initial iGEM research and assembling the team
13.12.2022
  • First presentation about “What is the iGEM?”
22.12.2022
  • Very first brainstorming team meeting - composition of the team at the beginning: Mateusz, Maciej, Marta, Marta, Aniela, Adam
06.01.2023
  • Brainstorming team meeting #2
10.01.2023
  • Brainstorming team meeting #3
17.01.2023
  • Brainstorming team meeting #4 - discussion about EDCs, Maks joins the team
22.01.2023
  • General team meeting #5 - project topic chosen - EDCs: phthalates
26.01.2023
  • General team meeting #6 - logo brainstorm

27.01.2023
  • Mini team meeting
30.01.2023
  • First team photos - meeting #7
01.02.2023
  • General team meeting #8
03.02.2023
  • Meeting with the Dean of the IFB - Presenting the idea of taking part in the iGEM 2023 competition to the faculty authorities
06.02.2023
  • General team meeting #9
11.02.2023
  • General team meeting #10
19.02.2023
  • Mini team meeting
20.02.2023
  • General team meeting #11

23.02.2023
  • General team meeting #12
  • design of fundraising materials
  • preparation of surveys for UG Open Days in March
01.03.2023
  • General team meeting #13
  • division of roles in the team
  • choice of the team name as IFB-Gdansk
13.03.2023
  • Erwin joins the team
15.03.2023
  • Meeting online with INSA Lyon1
15-16.03.2023
  • UG Open Days, conducting our first survey
17.03.2023
  • Talk with dr George di Cenzo
29.03.2023
  • General team meeting #14 - project presentation to our PI
05.04.2023
  • The great fundraising evening - reaching out to many institutions and companies
05-25.04.2023
  • Fundraising for the iGEM Registration Fee
18.04.2023
  • GFRG (Gdańska Fundacja Rozwoju Gospodarczego) - Our new sponsor
26.04.2023
  • General team meeting #15
27.04.2023
  • Our iGEM Registration Fee has been covered (3 days before the deadline)
29.04.2023
  • BioTeam Meeting #1
10.05.2023
  • BioTeam Meeting #2

11.05.2023
  • General team meeting #16
12.05.2023
  • Secondary PI joins the team
17.05.2023
  • BioTeam Meeting #3
19.05.2023
  • Talk with our PIs and prof. Michał Obuchowski

23.05.2023
  • BioTeam Meeting #4, Mikołaj and Julia join the team
  • Call with our Mentor - Gokul Bhaskaran
27.05.2023
  • Bowling - team integration
28.05.2023
  • BioTeam Meeting #5
  • Our stand at Fahrenheit Universities Science Picnic
31.05.2023
  • Meeting with our sponsor - BIOKOM
  • BioTeam Meeting #6 - planning our work according to the BioBricks standard
02.06.2023
  • BioTeam Meeting #7
05.06.2023
  • first meeting in cooperation with SPARK
  • Meeting with prof. Stanisław Ołdziej - talk about HPLC analysis and the possibility of using the equipment
06.06.2023
  • General team meeting #18

07.06.2023
  • Meeting with Igor Grochowina - talk about bioinformatics analysis of our enzymes
  • Meeting with our PI - Ph.D. Katarzyna Węgrzyn
08.06.2023
  • BioTeam Meeting #8

10.06.2023
  • Meeting with Maelys from INSA-ENS-Lyon iGEM Team

12.06.2023
  • Meeting with prof. Stanisław Ołdziej - HPLC Q&A
  • Meeting with SPARK #2
  • Our stand at 30-year anniversary of IFB
14.06.2023
  • iGEM StartUps: Biotech Venture Webinar
  • Our stand at IFB Student’s Day
16.06.2023
  • The Beyond Numbers Project: Reproducibility & Standardization Webinar
17.06
  • General team meeting #19
19.06.2023
  • Meeting with SPARK #3
  • Visit and workshops at Gdańskie Liceum Autonomiczne in Gdańsk
20.06.2023
  • BioTeam Meeting #9
  • Visit and workshops at III Liceum Ogólnokształcące im. Bohaterów Westerplatte in Gdańsk
24.06.2023
  • Meeting with our sponsor - BIOKOM
  • BioTeam Meeting #6 - planning our work according to the BioBricks standard
26.06.2023
  • General team meeting #20
  • Meeting with SPARK #4
02.07.2023
  • General team meeting #21
03.07.2023
  • Call with our Mentor - Gokul Bhaskaran
04.07.2023
  • Call with iGEM Lund Team

12.07.2023
  • BioTeam Meeting #10
15-16.07.2023
  • iGEM StartUps: Summer School
15.07.2023
  • Mini team meeting about wiki and hardware
16.07.2023
  • Mini team meeting about wiki
17.07.2023
  • Mini team meeting about Human Practices and Collaborations
20.07.2023
  • BioTeam Meeting #11
27.07.2023
  • Mini team meeting about our Social Media plans
  • Synthetic Biology in Space - Achievements and Possibilities Webinar

02.08.2023
  • Team Meeting about Social Media, Education and Human Practices plans for the rest of the competition time
03.08.2023
  • BioTeam Meeting #12
06.08.2023
  • BioTeam Meeting #13
07.08.2023
  • General team meeting #22
08.08.2023
  • Meeting with our PI - Ph.D. Katarzyna Węgrzyn
  • Call with Seoul Korea High School iGEM Team

09.08.2023
  • BioTeam Meeting #14
10.08.2023
  • General team meeting #23 - Wiki design ideas brainstorm
11.08.2023
  • Team Meeting and brainstorming ideas for Project Promotion Video
23.08.2023
  • BioTeam Meeting #15
  • Call with Ph.D. Magdalena Kulczycka - Director of BioForum - our sponsor
24.08.2023
  • Meeting with Marcel Thiel - HPLC learning
25.08.2023
  • Talk with Prof. Dr. Jörn Kalinowski

28.08.2023
  • Call with Thessaly iGEM Team

29.08.2023
  • Mini team meeting about wiki and hardware
30.08.2023
  • Call with BIOTON - our new sponsor
  • BioTeam Meeting #16
31.08.2023
  • Talk with Ph.D. Sylwia Klińska about Golden Gate Cloning Troubleshooting
07.09.2023
  • Mini team meeting about wiki

08.09.2023
  • Guest lecture in Pomeranian Science and Technology Park Gdynia
10.09.2023
  • General team meeting #25
12.09.2023
  • Mini meetings about wiki and education
13.09.2023
  • Call with Bioton representatives to talk about our project
14.09.2023
  • Meeting with SPARK #5
19.09.2023
  • Meeting with our PIs to discuss our last experiments
20.09.2023
  • Mini meeting about Human Practices wiki
21.09.2023
  • Visit and workshops at I Liceum Ogólnokształcące im. Ziemii Kujawskiej in Włocławek
22.09

Visit and workshops at:

  • V Liceum Ogólnokształcące im. Jana Pawła II in Toruń
  • IV Liceum Ogólnokształcące im. Tadeusza Kościuszki in Toruń
24.09.2023
  • General team meeting #26
27.09.2023
  • Our team visited Wastewater Treatment Plant in Toruń
02.10.2023
  • BioTeam Meeting #17 - wiki discussions
03.10.2023
  • Meeting with prof. Erwan Gueguen - PI of INSA Lyon1 iGEM 2022 Team

04.10.2023
  • organization of Pub Quiz

05.10.2023
  • BioTeam Meeting #17 - wiki discussions
06.10.2023
  • Our team visited Bioton facility in Macierzysz
06-12.10.2023
  • Full of intense wiki writing and designing