iGEM 2023 — Team ifb-gdansk

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### LABORATORY NOTEBOOK

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### 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**

## **Week #1**

**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**

## **Week #1**

**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.

## **Week #2**

**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).

## **Week #3**

**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.

## **Week #4**

**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**

## **Week #1**

**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.

## **Week #2**

**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.

## **Week #3**

**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.

## **Week #4**

**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.

## **Week #5**

**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**

## **Week #1**

**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.

## **Week #2**

**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.

## **Week #3**

**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 |

## **December 2022**

**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

## **January 2023**

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/1.png)

**27.01.2023**

* Mini team meeting

**30.01.2023**

* First team photos - meeting #7

## **February 2023**

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/2.jpeg)

**23.02.2023**

* General team meeting #12
* design of fundraising materials
* preparation of surveys for UG Open Days in March

## **March 2023**

**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

## **April 2023**

**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

## **May 2023**

**10.05.2023**

* BioTeam Meeting #2

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/3.jpeg)

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/4.jpg)

**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

## **June 2023**

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/5.jpeg)

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/6.jpeg)

**10.06.2023**

* Meeting with Maelys from INSA-ENS-Lyon iGEM Team

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/7.jpeg)

**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

## **July 2023**

**02.07.2023**

* General team meeting #21

**03.07.2023**

* Call with our Mentor - Gokul Bhaskaran

**04.07.2023**

* Call with iGEM Lund Team

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/8.png)

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/9.png)

## **August 2023**

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/10.png)

**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

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/11.jpeg)

**28.08.2023**

* Call with Thessaly iGEM Team

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/12.png)

**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

## **September 2023**

**07.09.2023**

* Mini team meeting about wiki

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/13.jpeg)

**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

## **October 2023**

**02.10.2023**

* BioTeam Meeting #17 - wiki discussions

**03.10.2023**

* Meeting with prof. Erwan Gueguen - PI of INSA Lyon1 iGEM 2022 Team

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/14.jpeg)

**04.10.2023**

* organization of Pub Quiz

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/15.jpeg)

**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

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

×
![]()

## **INTRODUCTION**

**Our main objective was to develop a novel biofilter capable of degrading health-hazardous phthalates.**

Dibutyl phthalate (DBP) and bis(2-ethylhexyl) phthalate (DEHP) belong to the group of phthalates that are hazardous to health, but which can be degraded by enzymes to easy degradable benzoic acid. Our idea is to use the tools of synthetic biology to obtain the enzymes esterase and decarboxylase. We have also prepared a protein scaffold with a modular structure that allows the immobilization of enzymes in various configurations and quantitative ratios to optimize the efficiency of the process. Cellulose inserts will be placed in a filter designed by us using 3D printing technology (Fig. 1).

![Educational timeline](https://static.igem.wiki/teams/4695/wiki/website/engineering/filtr-obszar-roboczy-1154-4x.png)

***Figure 1.** Schematic representation of a filter with a cellulose insert containing immobilized enzymes, conducting the biodegradation of phthalates into safe benzoic acid.*

In the process of implementing our project plan, we followed the engineering cycles approach to maximize our efficiency and increase the chances of engineering success. Initially, we collectively planned the next steps during meetings and discussions within the bio-team (design). Subsequently, we constructed individual components, bringing our ideas to life (build). To confirm that our objectives were met, we tested our systems (test), and the results allowed us to learn (learn) and acquire the knowledge necessary to improve elements of our project.

In our project, we identified six main cycles, all of which contributed to our success (Fig. 2):

1. DNA sequence design – basic and composite parts,
2. Preparation of the DNA distribution kit plate usable elements bank,
3. Obtaining the enzymes,
4. Obtaining the protein scaffold - scaffoldin,
5. Phthalate concentration analysis – HPLC,
6. Design and construction of a prototype filter.

The engineering cycles are detailed as follows:

![](https://static.igem.wiki/teams/4695/wiki/website/sponsors/cycle/pocz-tek-przyci-teschemat.svg)

[![](https://static.igem.wiki/teams/4695/wiki/website/sponsors/cycle/1-przyci-teschemat.svg)](#Header1)

[![](https://static.igem.wiki/teams/4695/wiki/website/sponsors/cycle/2-przyci-teschemat.svg)](#Header2)

[![](https://static.igem.wiki/teams/4695/wiki/website/sponsors/cycle/5-przyci-teschemat.svg)](#Header5)

[![](https://static.igem.wiki/teams/4695/wiki/website/sponsors/cycle/3-przyci-teschemat.svg)](#Header3)

[![](https://static.igem.wiki/teams/4695/wiki/website/sponsors/cycle/4-przyci-teschemat.svg)](#Header4)

![](https://static.igem.wiki/teams/4695/wiki/website/sponsors/cycle/koniec-przyci-teschemat.svg)

***Figure 2.** The diagram represents the plan of our project to construct a filter. Each number designates the distinct engineering cycles that accompanied us from the beginning to the achievement of our goal, which was the construction of a filter prototype.*

## **Table of contents**

[**Cycle 1: DNA sequence design – basic and composite parts**](#Header1)
[**Cycle 2: Preparation of the DNA distribution kit plate usable elements bank**](#Header2)
[**Cycle 3: Obtaining the enzymes**](#Header3)
[**Cycle 4: Obtaining the protein scaffold - scaffoldin**](#Header4)
[**Cycle 5: Phthalate concentration analysis – HPLC**](#Header5)

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **CYCLE 1: DNA SEQUENCE DESIGN – BASIC AND COMPOSITE PARTS**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

In this cycle, we designed the nucleotide sequences of the biological elements of our filter: enzymes and the immobilization protein scaffold. Our goal was to design a modular system, the expression of which could be carried out using the *"E. coli Protein Expression Toolkit,"* which is available on the iGEM DNA Distribution Kit Plates.

#### Iteration 1:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

The first and most time-consuming challenge for our team was the overall project planning. By reviewing the literature, we adapted a hypothetical enzymatic pathway for the degradation of phthalates, utilizing two enzymes from two different bacterial species. We selected two esterases and four decarboxylases that could potentially catalyze the chemical reactions we identified.

Simultaneously, we considered various methods for enzyme immobilization, such as binding to cellulose or encapsulation in alginate beads. We also contemplated the use of genetically modified bacterial cells for pollutant degradation.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We created a graphical design of the enzymatic pathway, connecting potentially useful enzymes from various publications (Fig. 3A). Taking inspiration from the Brno team (https://2020.igem.org/Team:Brno\_Czech\_Republic) and literature , we designed a protein scaffold in the form of a cellulosome (Fig. 3B).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-3-2.svg)

***Figure 3.** Graphical representation of our ideas*

a) The diagram represents the enzymatic pathway we utilized for the degradation of phthalates. In our project, we selected two esterases (PnbA, EstJ6) derived from Bacillus subtilis strain BJQ0005 and soil metagenomic libraries, and four decarboxylases (Dcx1, Dcx2, Dcx3, Dcx4) from Pantoea dispersa BJQ0007.

b) Structure of the cellulosome. which binds to cellulose fibers via CBD (cellulose binding domain) on one side, and on the other side binds enzymes that are in fusion with dockerins via cohesins.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We conducted numerous consultations of our ideas with specialists from various fields, including Professor Obuchowski, Professor Ołdziej and our advisors: Professor Czajkowski and Professor Węgrzyn.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

Together, we chose the cell-free path and a system that utilizes the cellulosome. We learned that during the design, we would have to consider aspects such as protein purification methods (e.g., adding a His-Tag) and a way to separate the dockerin from the enzymes (e.g., adding a sequence recognized by the TEV protease) to enable testing of native enzymes. Detailed project planning helped us streamline further processes, and having a general plan allowed us to proceed with its implementation.

#### Iteration 2:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

Once the details were established, we could proceed with designing sequences in the form of biobricks. Our aim was to maximize the modularity of our device so that we could utilize and exchange its elements, such as promoters or vectors, using the iGEM DNA Distribution Kit database. Given that the bacteria we will be working with are E. coli, we decided to prepare composite parts using the "E. coli Protein Expression Toolkit" (Table 1, Fig. 4).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

Table 2. Parts from iGEM Distribution kit plate we used in our project.

| Our name system | Registry ID part | Prefix and suffix | Length |
| --- | --- | --- | --- |
| Promoter | BBa\_J435350 | AB | 103 bp |
| Vector pUC | BBa\_J435330 | AF | 1769 bp |
| RBS | BBa\_J435385 | BC | 84 bp |
| linkerDT4 | BBa\_J435309 | DT4 | 29 bp |
| linkerDT4tev | BBa\_J435395 | DT4 | 35 bp |
| linkerEF | BBa\_J435361 | EF | 65 bp |
| linkerD4T5 | BBa\_J435349 | T4T5 | 38 bp |
| linkerT5Ehis | BBa\_J435369 | T5E | 27 bp |
| linkerDF | BBa\_J435393 | DF | 371 bp |
| pJUMP 23-1A | BBa\_J428347 | AF | 3113 bp |
| pJUMP 26-1A | BBa\_J428350 | AF | 2323 bp |
| pJUMP 28-1A | BBa\_J428353 | AF | 2520 bp |
| pJUMP 29-1A | BBa\_J428341 | AF | 2976 bp |
| GFP | BBa\_J435328 | CD | 722 bp |

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-4.svg)

***Figure 4.** . Assembly schematic of the E. coli expression toolkit. Parts in the E. coli expression toolkit (orange boxes) are flanked by Golden-Gate compatible fusion sites (grey boxes). Graphic based on: https://stanford.freegenes.org/products/expression-tookit#description*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We optimized the designed sequences in the OPTIMIZER program (http://genomes.urv.es/OPTIMIZER/) to ensure efficient expression in E. coli cells. We added appropriate suffixes and prefixes to the basic parts and then designed composite parts with elements from the "E. coli Protein Expression Toolkit" (Figure 5A and 5B). Before ordering synthetic sequences for basic parts, we conducted trial ligations in silico using the SnapGene program.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-3-2.svg)

***Figure 3.** Designed components for synthesis. a) Composite parts design of ScfL-His device (scaffoldin). b) Composite parts design of enzymes in fusion with dockerin (esterases: PnbA-DocScaB device, EstJ6-DocScaB device; decarboxylases: Dcx1-DocXylY device, Dcx2-DocXylY device, Dcx3-DocXylY device, Dcx4-DocXylY device).*

The diagram shows the elements of the 'E. coli Protein Expression Toolkit' (orange), the corresponding prefixes and suffixes (blue) and our designed basic parts (green).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We attempted to ligate the synthesized sequences with elements from the "E. coli Protein Expression Toolkit" using the Golden Gate method, but they did not yield the expected results. After attempting the ligation of our enzymes in fusion with the dockerin, we obtained results from restriction analysis that were inconsistent with our expectations. In the case of the scaffoldin, we did not observe protein production in the transformed bacteria. More details in engineering cycles: Obtaining the protein scaffold – scaffoldin, Obtaining the enzymes.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We determined that we need an alternative ligation method to Golden Gate, one that increases the chances of success and reduces the issues with matching the appropriate quantity of individual parts in the reaction mixture. We speculate that less complex reaction mixtures should lead to more efficient ligations.

#### Iteration 3:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

To streamline our work, we decided to combine the elements of our system through artificial DNA synthesis. Due to financial constraints, we could not order all the elements, so we created three constructs containing two esterases (PnbA-DocScaB device, EstJ6-DocScaB device) and one decarboxylase (Dcx1-DocXylY device) in fusion with dockerins.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We performed ligations of the ordered sequences with plasmids: vector pUC and pJUMP 23-1A, then transformed E. coli cells with these constructs.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We conducted restriction analyses and PCR (polymerase chain reaction) assays, which confirmed successful ligation and transformation. Furthermore, we were able to overproduce all three enzymes in E. coli cells. More details in engineering cycles: Obtaining the protein scaffold – scaffoldin, Obtaining the enzymes.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We successfully designed basic parts and then composite parts. The transformed bacteria carry plasmids containing sequences PnbA-DocScaB device and Dcx1-DocXylY device.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **CYCLE 2: PREPARATION OF THE DNA DISTRIBUTION KIT PLATE USABLE ELEMENTS BANK**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

In our project, we aimed to utilize elements we received as part of the iGEM distribution plate. To facilitate our work, we needed to prepare a cell bank containing the necessary elements: promoter ([BBa\_J435350](http://parts.igem.org/Part%3ABBa_J435350)), vectors ([BBa\_J435330](http://parts.igem.org/Part%3ABBa_J435330), [BBa\_J428347](http://parts.igem.org/Part%3ABBa_J428347), [BBa\_J428350](http://parts.igem.org/Part%3ABBa_J428350), [BBa\_J428353](http://parts.igem.org/BBa_J428353), [BBa\_J429341](http://parts.igem.org/Part%3ABBa_J429341)), RBS ([BBa\_J435385](http://parts.igem.org/Part%3ABBa_J435385)), linkers ([BBa\_J435309](http://parts.igem.org/Part%3ABBa_J435309), [BBa\_J435395](http://parts.igem.org/Part%3ABBa_J435395), [BBa\_J435349](http://parts.igem.org/Part%3ABBa_J435349), [BBa\_J435369](http://parts.igem.org/Part%3ABBa_J435369)), and terminator ([BBa\_J435361](http://parts.igem.org/Part%3ABBa_J435361)).

#### Iteration 1:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

In the first stage, we planned to transform E. coli cells with selected elements from the iGEM distribution plate kit. We chose the E. coli DH5α strain, which is well-regarded for plasmid overproduction.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We carried out the first transformation with the elements: promoter, vector pUC and RBS.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

After the transformation, we plated the cells on selective media containing kanamycin and ampicillin. We successfully obtained individual colonies containing the vector pUC (Fig. 6).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-6-wersja-2.jpg)

***Figure 6.** Cells plated on selective medium containing kanamycin and ampicillin after transformation with vector pUC part.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We successfully obtained cells containing the plasmid with vector pUC part. We learned that we should improve the method to increase the transformation efficiency.

#### Iteration 2:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We modified the basic transformation protocol by extending the incubation times of the samples and introducing a heat shock step.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We carried out another transformation, following the modified protocol, with the following elements: promoter and RBS.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We plated the cells after transformation on selective media containing ampicillin. Unfortunately, not a single bacterial colony grew.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We concluded that the method we chose, or the bacterial strain may not be suitable for these plasmids in this case.

#### Iteration 3:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We decided to test additional transformation methods, including electroporation, and used NEB® 10-beta Competent E. coli cells, which were transformed according to the producer's recommendations.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

As planned, we carried out further transformations with promoter and RBS parts.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

Once again, we plated the cells after transformation on selective media containing ampicillin. Unfortunately, as before, not a single bacterial colony grew.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We learned new transformation methods, but none of them yielded the expected results.

#### Iteration 4:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

During consultations with our advisor, we realized that we did not centrifuge the iGEM distribution kit plates before sampling the material. It was likely that the DNA was stuck to the walls, which resulted in a lower concentration in the obtained suspension than we had expected.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We repeated the transformation according to the producer’s instructions, using NEB10-beta competent cells. Before extracting genetic material, we centrifuged the iGEM distribution kit plate.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We plated the cells after transformation on selective media containing ampicillin. This time, we observed the presence of bacteria on all plates. We isolated the plasmids from them, and subsequent restriction analyses confirmed the presence of the desired parts (Fig. 7).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-7.svg)

***Figure 7.** Selected results of two restriction analyses. Plasmids from the iGEM distribution kit plate were transformed into bacteria, and the isolated plasmids were digested with the BsaI enzyme. The images show DNA separation in an agarose gel*

a) In the middle well, the band represents the isolated vector pUC. In the well on the right, the restriction enzyme-cut product is visible, with two bands corresponding to the speculated product lengths of the analysis (2120bp and 1773bp).

b) In the middle well is a plasmid containing the DT4 linker element. Three bands in this well correspond to plasmid conformations: supercoiled, circular, and linear. After restriction enzyme digestion (well on the right), only one plasmid conformation is visible - linear. The product of the restriction analysis has a length of 29 bp, so it is not visible in the gel.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

Plate centrifugation increased the amount of the product in the reaction mixture and allowed us to carry out successful transformations. This enabled us to prepare a cell bank containing plasmids with the parts necessary for the subsequent stages.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **CYCLE 3: OBTAINING THE ENZYMES**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

The aim was to overproduce protein-enzymes that we wanted to use in performing dibutyl phthalate (DBP) and bis(2-ethylhexyl) phthalate (DEHP) biodegradation reactions. In order to combine our enzymes with scaffoldin in the future, we prepared the design of enzyme-dockerin fusion proteins.

#### Iteration 1:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

Having synthesized the basic parts (see Cycle 1) and the plasmids containing the rest of the elements needed to construct the composite parts (see Cycle 2), we could proceed to the first ligation. We decided to place our elements on the vector pUC, which carries genes that determine resistance to two antibiotics: kanamycin and ampicillin. During ligation, the ampicillin resistance determinant element is cut out, so the sensitivity of the bacteria to this antibiotic is a selection factor for correct ligation and transformation.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We performed Golden Gate ligation and then transformed NEB® 10-beta Competent E. coli cells with the resulting mixture.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We seeded the transformed cells on selection medium containing kanamycin. We then made replicate colonies on ampicillin medium for negative selection (Fig. 8a). We selected bacterial colonies that did not grow on ampicillin medium, isolated their plasmids and performed restriction analysis (Fig. 8b).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-8.svg)

***Figure 8.** First BioBrick constructs based ligation testing.*

a) Bacterial colony replicates at the top cultured on kanamycin medium (positive selection), at the bottom cultured on ampicillin medium (negative selection). b) Gel photo after agarose gel electrophoresis of samples subjected to restriction analysis. On the left, simulated analysis in SnapGene software. After treating our devices with the restriction enzyme SapI, we did not observe cutting of the plasmid into two parts. The control samples are post-ligation mixtures.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

Restriction analysis showed that the bacteria that grew after transformation do not have the desired plasmids. We need a new method for ligation of the parts.

#### Iteration 2:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We decided to ligate elements of our expression system through DNA synthesis (see Cycle 1 iteration 3). We cloned the synthesized constructs: PnbA-DocScaB device, EstJ6-DocScaB device, Dcx1-DocXylY device into the commercial pBAD24 plasmid using the blunt-end ligation method.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

After ligation with the pBAD24 vector, we transformed E. coli Rosetta 2(DE3) cells.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We seeded the transformed cells onto the selection medium. We obtained post-transformation colonies potentially containing the constructs PnbA-DocScaB device and Dcx1-DocXylY device. We isolated plasmids from them and subjected them to restriction analysis, which did not yield results as expected (Fig. 9).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-9.svg)

***Figure 9.** Restriction analysis after ligation to the pBAD24 vector. The constructs PnbA-DocScaB device and Dcx1-DocXylY device were treated with EcoRI and EcoRV enzymes. As within the sequence of the constructs there is one site recognized by the EcoRI enzyme, we expected to observe one band on the gel. EcoRV enzyme was used to perform ligation, so after ligation there should be no sites recognized by the enzyme within the sequence.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

The method using blunt ends is inefficient for chosen plasmids.

#### Iteration 3:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We decided to try GoldenGate ligation to the pUC vector once again, this time having the devices assembled by DNA synthesis (see Cycle 1 iteration 3).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

Having the elements ligated using DNA synthesis, we performed their ligation with the vector pUC using the GoldenGate method. Then we carried out the transformation using NEB10-beta competent cells.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We seeded the transformed cells onto the selection medium containing ampicillin. We obtained transformed colonies potentiated with the PnbA-DocScaB device, EstJ6-DocScaB device and Dcx1-DocXylY device constructs. We isolated plasmids from them and subjected them to restriction analysis, which this time gave us expected results (Figure 10).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-10.svg)

***Figure 10.** Gel obtained by agarose electrophoresis showing the result of restriction analysis. After ligation, the constructs lose sequences recognized by the BsaI enzyme (no cut - one band), while they gain sequences recognized by SapI (cut - two bands).*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

Restriction analysis confirmed that the cells carry a plasmid containing our constructs. We also learned that the pUC vector is high-copy and is not suitable for overproduction of all proteins.

#### Iteration 4:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We re-examined the iGEM distribution kit plate, where we found plasmids belonging to the JUMP collection. We identified 4 potential vectors that we could use: pJUMP 23-1A, pJUMP 26-1A, pJUMP 28-1A, pJUMP 29-1A. Plasmids, according to the description, were characterized by different copy number , but we decided to confirm it.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We performed bacterial transformation with all four selected vectors.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We seeded the transformed bacteria onto a selection medium containing kanamycin. We transferred the resulting colonies to liquid medium and cultured them overnight. Under UV light, we analyzed the fluorescence intensity of our cultures (Figure 11), which correlated with plasmid copy number.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-11.jpg)

***Figure 11.** Photo of cultures containing cells transformed with the following plasmids: pJUMP23-1A, pJUMP 26-1A, pJUMP 28-1A and pJUMP 29-1A. Photo was taken while the tubes were illuminated with UV light.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

For further studies, we selected plasmid pJUMP23-1A, which had the lowest copy number , as confirmed by the lowest fluorescence intensity among the plasmids tested.

#### Iteration 5:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

The next step in our project was to ligate the constructs PnbA-DocScaB device, EstJ6-DocScaB device and Dcx1-DocXylY device with the vector pJUMP23-1A.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We ligated our constructs with the pJUMP23-1A vector using the GoldenGate method and then transformed E. coli BL21 cells with the resulting ligation mixture.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We seeded the transformed bacteria onto a selection medium containing kanamycin. None of the bacterial colonies that grew fluoresced under UV light, which preliminarily suggested that ligation was successful and the correct part of the plasmid was excised. We then isolated the plasmids and performed restriction analysis (Fig 12a). We also performed PCR reactions analysis to confirm the presence of all elements of the construct on the isolated plasmid containing the PnbA-DocScaB device. (Fig 12b).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-12.svg)

***Figure 12.** a) Gel obtained by agarose electrophoresis showing the result of restriction analysis with enzymes: BsaI and XbaI. After ligation, the constructs lose sequences recognized by the BsaI enzyme (no cut - one band). All our devices have two sequences recognized by XbaI (cut - two bands). b) Agarose gel with PCR products using primers complementary to the sequences of all biobricks used to create the PnbA-DocScaB device construct. On the left, visible simulation performed in SnapGene software. The control sample was the post-reaction mixture samples without DNA template.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

Restriction analysis showed that we were able to obtain plasmids containing 2 of the 3 enzymes: pJUMP23-1A-Dcx1-DocXylY and pJUMP23-1A-PnbA-DocScaB. In addition, PCR analysis showed that the pJUMP23-1A-PnbA-DocScaB construct contains all the necessary elements.

#### Iteration 6:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

At this stage, we could move on to try to overproduce our enzymes.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We carried out protein overproduction by adding IPTG (Isopropyl β-d-1-thiogalactopyranoside) to cell cultures containing Dcx1-DocXylY and PnbA-DocScaB elements on pJUMP23-1A plasmid.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

After induction, we collected culture samples and resuspended them in Laemmli buffer (a sample buffer for denaturing and loading of protein samples). We subjected the samples to polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) to quickly check whether protein overproduction had occurred (Fig. 13).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-13.svg)

***Figure 13.** Gel obtained by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Arrows indicate proteins that were significantly increased. The predicted mass of dockerin-esterase (PnbA-DocScaB) is 64.6 kDa, while that of dockerin-decarboxylase (Dcx1-DocXylY) is 28.6 kDa.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We managed to overproduce proteins: esterase and decarboxylase.

#### Iteration 7:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

The final step in this cycle was the purification of the resulting protein - PnbA-DocScaB. Our proteins had a His-Tag, so we chose Immobilized Metal Affinity Chromatography (IMAC) for purification.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We lysed the cells using sonication and then separated the proteins on a Ni-NTA resin column. After washing the bed with buffer, we recovered the proteins bound to the bed by washing with an imidazole gradient.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We visualized the fractions obtained by washing and protein recovery using SDS-PAGE (Fig. 14).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-14.svg)

***Figure 14.** Gel obtained by SDS-PAGE after purification of proteins. We can see the sample applied to the column (supernatant load), the fraction not bound to the bed (flow through), selected wash fractions (11,12,14) and selected fractions extracted from imidazole gradient protein recovery (5, 10, 12, 15, 18, 21, 24, 27).*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We were able to obtain a purified enzyme (esterase) in fusion with dockerin: PnbA-DocScaB. We were able to proceed to enzyme assays, which are shown in Cycle 5.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **CYCLE 4: OBTAINING THE PROTEIN SCAFFOLD - SCAFFOLDIN**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

The goal of this cycle was to overproduce a protein scaffold - scaffoldin. This element, by binding to cellulose fibers, would enable us to immobilize enzymes.

#### Iteration 1:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We first wanted to obtain bacteria carrying a plasmid containing scaffoldin: ScfL. For this purpose, we decided to ligate selected elements from iGEM distribution kit plate with our protein, thus obtaining ScfL-His device.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We first performed the ligation of scaffoldin (ScfL) with iGEM distribution kit plate elements such as a promoter, RBS, terminator, linkers, and vector pUC. We performed the ligation according to the Golden Gate protocol.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We then cultured the bacteria onto a positive selection medium containing kanamycin, and then prepared replicates on a negative selection medium containing ampicillin (after successful ligation, the bacteria should be sensitive to this antibiotic). We isolated plasmids from selected colonies and performed restriction analysis.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-15.svg)

***Figure 15.** Gel obtained by agarose electrophoresis showing the result of restriction analysis. After ligation, the constructs gain sequences recognized by SapI (cut - two bands). The control sample was a ligation mixture. On the left, visible simulation performed in SnapGene software.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

Restriction analysis showed that bacteria that grew after transformation lacked the desired plasmid.

#### Iteration 2:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We decided to repeat the ligation using the GoldenGate method but making a few modifications to the protocol. We also decided to use a different vector, this time choosing pJUMP23-1A.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We modified the previously used GoldenGate ligation protocol by increasing the amount of ligase and the volume of the reaction mixture, as well as increasing the duration of the ligation cycles. The ligation mixture was then used to transform E. coli BL21 cells.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

Transformed cells were cultured onto a selection medium containing kanamycin. We selected colonies that did not fluoresce under UV light, isolated plasmids from them and performed restriction analysis (Fig. 16).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-16.svg)

***Figure 16.** Gel obtained by agarose electrophoresis showing the result of restriction analysis. After ligation, the constructs lose sequences recognized by the BsaI enzyme (we predict if no cut - one band), while they gain sequences recognized by SapI (we predict if cut - two bands).*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

We were unable to confirm by restriction analysis that the bacteria carry a plasmid containing ScfL-His device.

#### Iteration 3:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

We decided to repeat the ligation for the third time, making further modifications to the GoldenGate ligation protocol. We also planned different testing methods this time because we wanted to make sure that in the case of scaffoldin, the results of the restriction analysis were not false negatives.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We modified the GoldenGate ligation protocol used by increasing the number of ligation cycles (from 30 to 60). We also increased the amount of the restriction enzyme and ligase. We then transformed E. coli NEB 10beta cells with the ligation mixture.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We modified the GoldenGate ligation protocol used by increasing the number of ligation cycles (from 30 to 60). We also increased the amount of the restriction enzyme and ligase. We then transformed E. coli NEB 10beta cells with the ligation mixture.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-17.svg)

***Figure 17.** ScfL-His device ligation correctness test. A) Gel obtained by SDS-PAGE after induction of scaffoldin overproduction. We can observe thickened bands in control samples corresponding to PnbA-DocScaB (64.6 kDa), Dcx1-DocXylY (28.6 kDa) and GFP [fluorescent colony] (26.9 kDa). For research samples, we did not observe significant differences at the height corresponding to scaffoldin mass (77,8 kDa). b) Gel obtained by agarose electrophoresis of the PCR reaction products. On the left, visible simulation performed in SnapGene software. Five of the seven analyzed reaction products are in a different location than predicted.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

The results of our tests were inconclusive. In the case of SDS-PAGE, perhaps protein production was too low to detect with our method. In the PCR analysis, only two products were in the correct location, so perhaps ligation did not occur properly. In the case of scaffoldin, we ran out of time to complete the research.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **CYCLE 5: PHTHALATE CONCENTRATION ANALYSIS – HPLC**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

The main goal of this cycle was to analyse the enzymatic activity of our proteins. Due to the time limit, we managed to test the activity of esterase in fusion with dockerin (PnbA-DocScaB). We used high-performance liquid chromatography (HPLC) to conduct the analyses.

#### Iteration 1:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

In order to measure the concentrations of substrates and products of enzymatic reactions [i.e., dibutyl phthalate (DBP), monobutyl phthalate (MBP), butyl benzoate (BB), phthalic acid (PA), benzoic acid (BA)] by high-performance liquid chromatography (HPLC), we had to plot a calibration curve.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

We prepared serial dilutions of the analyzed HPLC standards in a 20% solution of acetonitrile in phosphate-buffered saline (PBS). The samples were then loaded onto a chromatography column and separated for 1 hour.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We plotted calibration curves based on the chromatography results (Fig. 18).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-18.svg)

***Figure 18.** Calibration curves of substrates and products of enzymatic reactions i.e., dibutyl phthalate (DBP), monobutyl phthalate (MBP), butyl benzoate (BB), phthalic acid (PA), benzoic acid (BA).*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

alibration curves plotted for PA, MBP and BA on the first chromatograph have the coefficient of determination (R2) above 0,99 giving a reasonably good fit. Calibration curves plotted for DBP and BB, on the other hand, have quite low R2, therefore values calculated using them would not be very reliable.

#### Iteration 2:

---

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/1-design-cykl.svg)

## DESIGN

After obtaining the purified PnbA-DocScaB and standard curves, we were able to move on to testing phthalate degradation activity.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/2-build-cykl.svg)

## BUILD

The analyses of enzymatic activity were done in 20% ACN in PBS to increase substrate solubility. The reaction was carried out for 30 minutes at 37°C. We tried dibutyl phthalate (DBP) and butyl benzoate (BB) as substrates.

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/3-test-cykl.svg)

## TEST

We used high-performance liquid chromatography to conduct the analyses (Fig. 19).

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/expandbig.svg)

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/figure-19.svg)

***Figure 19.** Diagrams prepared based on the results of HPLC analysis. Control samples did not contain esterase. a) Mean dibutyl phthalate concentration in samples – substrate of the reaction. b) Mean monobutyl phthalate concentration in samples – product of the reaction. c) Mean butyl benzoate concentration in samples – substrate of the reaction. d) Mean benzoic acid concentration in samples – product of the reaction.*

![](https://static.igem.wiki/teams/4695/wiki/website/engineering/4-learn-cykl.svg)

## LEARN

For the degradation reaction of the substrates (dibutyl phthalate and butyl benzoate) in the test sample, we can observe a significant increase in the concentration of the degradation reaction product (monobutyl phthalate and benzoic acid, respectively).

Unfortunately, the substrate concentration changed significantly after 30 minutes in both the control and test sample. These unexpected changes in substrate concentration may be related to the change in temperature during incubation, since DBP and BB did not form a homogeneous solution in 20% ACN in PBS, so an increase in temperature may have altered the substrate distribution between phases.

In conclusion, although this experiment did not provide data on reaction kinetics and enzyme activity, it still confirms that PnbA-DocScaB catalyzes the hydrolysis of ester bonds in MBP and BB at room temperature.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

×
![]()

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-1-scheme.svg)

***Figure 1.** General plan of our biological project. In blue are the plans we have managed to fulfill
 and in red are the parts of the plan for which we did not have enough time. Two-color boxes have been partially done.*

## **INTRODUCTION**

Our dream is to construct a biological filter that would degrade phthalic acid esters (PAE), removing these hazardous compounds from water. We have found enzymes that together would transform PAE into benzoic acid and designed a method of immobilizing them to cellulose, using a structure adapted from cellulosome - a protein complex used by cellulolytic bacteria. To achieve our goal, we have ordered protein-coding sequences from Integrated DNA Technologies, Inc. (IDT). Then we cloned them to the expression vectors. We have managed to overproduce two of them and purify one of them. This one enzyme is PnbA-DocScaB - a fusion protein with an enzymatic domain of p-nitrobenzyl esterase from *Bacillus subtilis BJQ0005* (locus tag GTW28\_17760) (Xu Youqiang, 2021) and a dockerin domain DocScaB. We confirmed that our fusion protein retains the ability to catalyze ester bond hydrolysis at least in dibutyl phthalate (DBP) and butyl benzoate (BB) case.

In table 1., there are all the new parts that we have gathered and have added to the iGEM parts registry. They are mentioned later in the description of the experiments we have conducted.

Table 1. List of parts registered by our team

|  | | Bba code | | | Individual code |
| --- | --- | --- | --- | --- | --- |
|  | Part name | BBa\_KXXXxxxx | BBa\_KxxxXXxx | BBa\_KxxxxxXX |
| Basic parts | pnbA | BBa\_K469 | 50 | 10 | [BBa\_K4695010](http://parts.igem.org/Part%3ABBa_K4695010) |
| estJ6 | 11 | [BBa\_K4695011](http://parts.igem.org/Part%3ABBa_K4695011) |
| dcx1 | 20 | [BBa\_K4695020](http://parts.igem.org/Part%3ABBa_K4695020) |
| dcx2 | 21 | [BBa\_K4695021](http://parts.igem.org/Part%3ABBa_K4695021) |
| dcx3 | 22 | [BBa\_K4695022](http://parts.igem.org/Part%3ABBa_K4695022) |
| dcx4 | 23 | [BBa\_K4695023](http://parts.igem.org/Part%3ABBa_K4695023) |
| scfL | 30 | [BBa\_K4695030](http://parts.igem.org/Part%3ABBa_K4695030) |
| docXylY | 40 | [BBa\_K4695040](http://parts.igem.org/Part%3ABBa_K4695040) |
| docScaB | 41 | [BBa\_K4695041](http://parts.igem.org/Part%3ABBa_K4695041) |
| docCel48A | 42 | [BBa\_K4695042](http://parts.igem.org/Part%3ABBa_K4695042) |
| Composite parts | pnbA-DocScaB | 51 | 10 | [BBa\_K4695110](http://parts.igem.org/Part%3ABBa_K4695110) |
| estJ6-DocScaB | 11 | [BBa\_K4695111](http://parts.igem.org/Part%3ABBa_K4695111) |
| dcx1-DocXylY | 20 | [BBa\_K4695120](http://parts.igem.org/Part%3ABBa_K4695120) |
| dcx2-DocXylY | 21 | [BBa\_K4695121](http://parts.igem.org/Part%3ABBa_K4695121) |
| dcx3-DocXylY | 22 | [BBa\_K4695122](http://parts.igem.org/Part%3ABBa_K4695122) |
| dcx4-DocXylY | 23 | [BBa\_K4695123](http://parts.igem.org/Part%3ABBa_K4695123) |
| scfL-His | 30 | [BBa\_K4695130](http://parts.igem.org/Part%3ABBa_K4695130) |
| pnbA-DocScaB device | 52 | 10 | [BBa\_K4695210](http://parts.igem.org/Part%3ABBa_K4695210) |
| estJ6-DocScaB device | 11 | [BBa\_K4695211](http://parts.igem.org/Part%3ABBa_K4695211) |
| dcx1-DocXylY device | 20 | [BBa\_K4695220](http://parts.igem.org/Part%3ABBa_K4695220) |
| dcx2-DocXylY device | 21 | [BBa\_K4695221](http://parts.igem.org/Part%3ABBa_K4695221) |
| dcx3-DocXylY device | 22 | [BBa\_K4695222](http://parts.igem.org/Part%3ABBa_K4695222) |
| dcx4-DocXylY device | 23 | [BBa\_K4695223](http://parts.igem.org/Part%3ABBa_K4695223) |
| scfL-His device | 30 | [BBa\_K4695230](http://parts.igem.org/Part%3ABBa_K4695230) |

## **Table of contents**

[**Obtaining plasmids from iGEM Distribution for later cloning**](#Header1)
[**Obtaining bacteria able to express ScfL using Golden Gate assembly of multiple parts**](#Header2)
[**Obtaining bacteria producing enzymes by blunt-end cloning**](#Header3)
[**Obtaining bacteria producing enzymes (vector pUC)**](#Header4)
[**Screening of pJUMP vectors**](#Header5)
[**Obtaining bacteria producing enzymes (pJUMP23-1A)**](#Header6)
[**Comparison of PnbA-DocScaB, EstJ6-DocScaB and Dcx1-DocXylY protein overproduction**](#Header7)
[**Obtaining purified PnbA-DocScaB and Dcx1-DocXylY**](#Header8)
[**Obtaining native PnbA by TEV cleavage**](#Header9)
[**Analysis of the thermostability of PnbA-DocScaB on Tycho (NanoTemper)**](#Header10)
[**Plotting a calibration curve of standards on HPLC**](#Header11)
[**Analysis of PnbA-DocScaB activity against DBP and BB**](#Header12)

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **OBTAINING PLASMIDS FROM IGEM DISTRIBUTION FOR LATER CLONING**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

We needed to amplify the sequences required for the ligation of our constructs (listed below) which were provided to us in iGEM Distribution. These sequences were in pre-cloned form, so we decided to transform them to Escherichia coli strains DH5α and NEB® 10-β and isolate them in higher concentrations viable for cloning. To examine if obtained plasmids contain the desired sequences, we made a restriction analysis by cutting these plasmids with BsaI, which was supposed to cut out sequences of interest. We then separated them using agarose gel electrophoresis.

## **EXECUTION**

1

#### Competent cells preparation

[Competent cell preparation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/1.pdf)

2

#### Transformation of *E. coli*

---

Optimization of this step was time-consuming. Because of that, we ended up transforming some of the parts to *E. coli* strain DH5α and some to NEB® 10-β. Most of the parts transformed to DH5α were transformed using the protocol included below, although sometimes there were some changes when we were trying to optimize the method. Additionally, the promoter was transformed using the protocol for electroporation. Parts transformed to NEB® 10-β were transformed using the protocol included below.

Table 2. Parts from iGEM Distribution we used:

| Our name system | Registry ID part | Prefix and suffix | Length | Concentration (ng/µl) | *E. Coli* strain transformed |
| --- | --- | --- | --- | --- | --- |
| Promoter | BBa\_J435350 | AB | 103 bp | 17.5 / 22\* | DH5α |
| Vector pUC | BBa\_J435330 | AF | 1769 bp | 45.3 | DH5α |
| RBS | BBa\_J435385 | BC | 84 bp | 47.6 | NEB® 10-β |
| linkerDT4 | BBa\_J435309 | DT4 | 29 bp | 34.4 | NEB® 10-β |
| linkerDT4tev | BBa\_J435395 | DT4 | 35 bp | 39 | NEB® 10-β |
| linkerEF | BBa\_J435361 | EF | 65 bp | 45.3 | NEB® 10-β |
| linkerD4T5 | BBa\_J435349 | T4T5 | 38 bp | 32 | NEB® 10-β |
| linkerT5Ehis | BBa\_J435369 | T5E | 27 bp | 65.4 | NEB® 10-β |
| GFP | BBa\_J435328 | CD | 722 bp | 21.mar | DH5α |
| linkerDF | BBa\_J435393 | DF | 371 bp | 55.9 | NEB® 10-β |

\*Plasmid isolated twice

[Protocol for electroporation](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/5.pdf)

[Transformation protocol for *E. coli* strain DH5α](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/9.pdf)

[Transformation protocol for NEB® 10-β](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/3.pdf)

3

#### Isolation of plasmid

---

We inoculated 5 ml of LB with 100 µg/ml ampicillin with a transformed colony of each part. Afterwards we isolated the DNA from the liquid culture. Isolated DNA was suspended in NF water and later used for ligation. This step was performed using the A&A Plasmid Mini Kit for plasmid isolation.

[Protocol for plasmid isolation](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/4.pdf)

4

#### BsaI restriction testing on isolated plasmids

---

We treated each isolated plasmid with BsaI, an enzyme used in Golden Gate assembly, which should result in cutting the adequate part from the plasmid. We added 5 µl of isolated plasmid to the reaction mixture. Next, the reaction products were separated in 1%, 0.5 x TBE agarose gel electrophoresis with a constant voltage of 100 V. We used 5 µl of NEB Purple 1 kb+ ladder as marker and A&A 6x loading buffer mixed with GelRed to achieve a final concentration of 0,6%.

[Protocol for plasmid isolation](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/6.pdf)

5

#### Plasmid concentration measurement

---

The concentration of each isolated plasmid was measured using Nanodrop.

[Protocol for Nanodrop](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/7.pdf)

## RESULTS

Gel electrophoresis results:

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-2-distribution-rest-1.svg)

***Figure 2.** Gel with restriction analysis of vector pUC, promoter, RBS, GFP and linkerDF*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-3-distribution-rest-2.svg)

***Figure 3.** . Gel with restriction analysis of linkerDT4, linkerDT4tev, linkerT4T5, linkerT5E, linkerEF*

Vector pUC and GFP sequences are long, so they could be observed as bands on the gel in figure 3. when cut with BsaI. Promoter, RBS and linkers are too short to be seen on the gel. We can deduce that BsaI cleavage occurred only because of the presence of a smear under main bands and the absence or fading of the 3 plasmid forms (linear, open circular and supercoiled), since after restriction mainly linear DNA should remain. However, it cannot be excluded that the smear might have been a result of using a greater sample volume than in the case of the uncut plasmids.

DNA concentration measurement results are presented in the Table 2.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **OBTAINING BACTERIA ABLE TO EXPRESS SCFL USING GOLDEN GATE ASSEMBLY OF MULTIPLE PARTS**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

Initially, we wanted to assemble all the expression vectors for overexpression of one esterase PnbA-DocScaB, four decarboxylases – Dcx1-DocXylY, Dcx2-DocXylY, Dcx3-DocXylY and Dcx4-DocXylY and scaffoldin ScfL, using Golden Gate assembly of all necessary parts (promoter, RBS, CDS, vector backbone and linkers). Later, we decided to order synthetized expression devices: PnbA-DocScaB device, EstJ6-DocScaB device (an esterase we included to the project later) and Dcx1-DocXylY device and use them instead. We could not afford to order a sequence as big as a scaffoldin with all the necessary regulatory sequences. Therefore, we tried to optimize Golden Gate assembly to obtain the expression vector of scaffoldin from following parts: Promoter ([BBa\_J435350](http://parts.igem.org/Part%3ABBa_J435350)), RBS ([BBa\_J435385](http://parts.igem.org/Part%3ABBa_J435385)), Scaffoldin CDS ([BBa\_K4695030](http://parts.igem.org/Part%3ABBa_K4695030)), linkerDT4 ([BBa\_J435309](http://parts.igem.org/Part%3ABBa_J435309)), linkerT4T5 ([BBa\_J435349](http://parts.igem.org/Part%3ABBa_J435349)), linkerT5Ehis ([BBa\_J435369](http://parts.igem.org/Part%3ABBa_J435369)), linkerEF ([BBa\_J435361](http://parts.igem.org/Part%3A)) and vector. In the third and final iteration, we used pJUMP 23-1A ([BBa\_J428347](http://parts.igem.org/Part%3A)) as a vector backbone. For more information about previous cloning attempts, check the engineering cycle page.

Right after transformation, we did not yet have the PCR primers, so we could not check if transformed bacteria contain the desired sequences. Instead, we looked for transformants overproducing a protein with molecular weight equal to scaffoldin, namely 79 kDA.

Later on, we obtained primers allowing for verification of cloning with PCR and conducted the analysis. First, we did colony-PCR looking for just the ScfL CDS. Then we chose the best candidates and performed 14 PCR reactions with the following primer pairs:

* fJUMP\_1\_F + pJUMP\_1\_R
* fJUMP\_1\_F + RBS\_348\_R
* fJUMP\_1\_F + Scaf\_R
* fJUMP\_1\_F + DT4\_274\_R
* fJUMP\_1\_F + T5E\_332 \_R
* fJUMP\_1\_F + Term\_324\_R
* Prom\_296\_F + pJUMP\_1\_R

Every pair was used in two reactions: one with template DNA and one without template DNA as a negative control. Sequences complementary to the primers are shown in Fig. 4. This way, we wanted to verify if all the parts assembled correctly.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-4-scfl-map.svg)

***Figure 4.** . Scheme of pJUMP23-1A-scfL expression vector with primers used for verification of construct. The position of primers is shown in purple.*

## **EXECUTION**

1

#### Competent *E. coli* strain BL21(DE3) cells preparation

---

We used competent cells prepared beforehand by Julia Karasińska for another project and kept frozen at -80 °C.

2

#### Golden Gate assembly

---

After consulting our initial problems with dr Sylwia Klińska, we modified the NEB Golden Gate assembly protocol

Deviations from the protocol: We extended the reaction to 60 cycles, 10 minutes each – 10 hours total. We used 1:1 molar ratio for each part. We increased total reaction volume to 25 µl and increased the buffer concentration to 1,2X. We conducted the reaction in two versions: in one we added 1.5 µl of BsaI and 0,5 µl of T4 ligase, and in the other, we added 3 µl of BsaI and 1 µl of T4 ligase.

[Golden Gate assembly protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/8.pdf)

3

#### Transformation of *E. coli* strain BL21(DE3)

---

pJUMP23-1A has sfGFP gene flanked by BsaI recognized sequences. Due to this, bacteria harboring unmodified plasmid fluoresce and those harboring vectors with desired sequence cloned into them lack the ability to fluoresce. This allowed for fast and simple recognition of colonies that were transformed with the unmodified vector. We chose *E. coli* strain BL21 (DE3) as a host for our vector because it is a strain tailored for protein synthesis.

[Transformation protocol for *E. coli*  strain BL21(DE3)](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/9.pdf)

4

#### SDS-PAGE screening for ScfL production after IPTG induction

---

At this point, we did not yet have PCR primers, so we could not check if the transformed bacteria contained desired sequences. Instead, we looked for transformants overproducing a protein with molecular weight equal to scaffoldin, namely 79 kDA.

We transferred 9 colonies from cloning with extra enzymes and 9 colonies from cloning with fewer enzymes, only non-fluorescent ones, onto LB-agar with 1 mM IPTG and kept them overnight at 37 °C. The next day, we picked a colony from each transformant transferred, lysed them in Laemmli buffer and separated in SDS-PAGE.

We did this twice, but the second time we took 14 random non-fluorescent colonies from step 3 transformation, one fluorescent colony as negative control, one colony expressing PnbA-DocScaB and one colony expressing Dcx1-DocXylY as positive controls and inoculated 5 ml of LB with 30 µg/µl with them. Additionally, one non-fluorescent colony was inoculated in duplicate. After 2.5 hours of incubation at 37 \\ with 120 RPM shaking, 5 µl of 1 M IPTG was added to the cultures, except for the duplicate of a non-fluorescent colony, and left overnight.

Next day, OD600 of the cultures was checked. To consider these differences, we calculated the amount of culture used for the analysis using the formula 1 ml / OD600. The calculated amounts were centrifuged for 5 minutes with 8000 RCF. Pellet was lysed in 50 µl of Laemmli buffer and used in SDS-PAGE.

Deviations from the protocol: Gel was separated at a constant voltage of 180 V. Destaining solution treatment was skipped.

[SDS-PAGE protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/13.pdf)

5

#### Colony-PCR screening

---

50 colonies from step 3 were numbered. Colonies were grouped into fives. For each five, samples were taken from the colonies and mixed together in 20 µl of nuclease-free (NF) water. Samples were then incubated at 95 °C for 10 minutes, after which they were centrifuged briefly at 10 000 RCF. 2 µl of each sample was mixed with 1,25 µl of 10 µM forward primer fJUMP\_1\_F and 1,25 µl of 10 mM reverse primer Scaf\_R, 12.5 µl of NEB Q5® High-Fidelity 2X Master Mix and 8 µl of NF water. PCR products were separated in 2% 0,5xTBE agarose gel electrophoresis with a voltage of 100V. Additionally we prepared two controls – one without template DNA and the other without primers.

We used colonies 11, 12, 13, 14 and 15 the second time (all being the result of ligation with extra enzymes), as their band was the thickest. We performed colony-PCR again in the same manner, except every colony was prepared separately this time. PCR products were separated in 1% 0.5xTBE agarose gel electrophoresis with a voltage of 100 V. We used 5 µl of NEB Purple 1kb+ ladder as a marker and A&A 6x loading buffer mixed with GelRed to a final concentration of 0.6%.

Tab. 3. PCR program

|  | Initial denaturation | Denaturation | Annealing | Elongation | Final elongation | Hold |
| --- | --- | --- | --- | --- | --- | --- |
| Temperature | 98°C | 98°C | 65°C | 72°C | 72°C | 10°C |
| Time [s] | 30 s | 10 s | 30 s | 80 s | 120 s | indefinite |
| Number of repeats | 1 | 30x |  |  | 1 | 1 |

6

#### Isolation of plasmid

---

5 tubes with 5 ml of LB + 30 µg/ml kanamycin were inoculated with colonies 11, 12, 13, 14 and 15 and kept at 37 °C with shaking overnight.

[Plasmid isolation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/4.pdf)

7

#### Confirmation of proper assembly by PCR

---

14 PCR tubes were filled as shown in table 4. Reagents were mixed on ice and centrifuged briefly after mixing. The thermocycler was programmed according to table 5. PCR products were separated in 1% 0,5xTBE agarose gel electrophoresis with a voltage of 100 V. We used 5 µl of NEB Purple 1kb+ ladder as marker and A&A 6x loading buffer mixed with GelRed to a final concentration of 0,6%. Additionally, we prepared a control without template DNA.

Tab. 4.

| No. | Forward primer | Reverse primer | Template [µl] | Master Mix° [µl] | NF water [µl] |
| --- | --- | --- | --- | --- | --- |
| 1 | fJUMP\_1\_F | pJUMP\_1\_R | 1 | 12.5 | 9 |
| 2 | fJUMP\_1\_F | pJUMP\_1\_R | 0 | 12.5 | 10 |
| 3 | fJUMP\_1\_F | RBS\_348\_R | 1 | 12.5 | 9 |
| 4 | fJUMP\_1\_F | RBS\_348\_R | 0 | 12.5 | 10 |
| 5 | fJUMP\_1\_F | Scaf\_R | 1 | 12.5 | 9 |
| 6 | fJUMP\_1\_F | Scaf\_R | 0 | 12.5 | 10 |
| 7 | fJUMP\_1\_F | DT4\_274\_R | 1 | 12.5 | 9 |
| 8 | fJUMP\_1\_F | DT4\_274\_R | 0 | 12.5 | 10 |
| 9 | fJUMP\_1\_F | T5E\_332\_R | 1 | 12.5 | 9 |
| 10 | fJUMP\_1\_F | T5E\_332\_R | 0 | 12.5 | 10 |
| 11 | fJUMP\_1\_F | Term\_324\_R | 1 | 12.5 | 9 |
| 12 | fJUMP\_1\_F | Term\_324\_R | 0 | 12.5 | 10 |
| 13 | Prom\_296\_F | pJUMP\_1\_R | 1 | 12.5 | 9 |
| 14 | Prom\_296\_F | pJUMP\_1\_R | 0 | 12.5 | 10 |

Tab. 5. PCR program

|  | Initial denaturation | Denaturation | Annealing | Elongation | Final elongation | Hold |
| --- | --- | --- | --- | --- | --- | --- |
| Temperature | 98°C | 98°C | 65°C | 72°C | 72°C | 10°C |
| Time | 30 s | 10 s | 30 s | 80 s | 120 s | indefinite |
| Number of repeats | 1x | 30x | | | 1x | 1x |

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-5-colony-page-1.svg)

***Figure 5.** Gel from SDS-PAGE screening for ScfL production after IPTG induction on solid medium, gel 1.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-6-colony-page-2.svg)

***Figure 6.** . Gel from SDS-PAGE screening for ScfL production after IPTG induction on solid medium, gel 2.*

We have not prepared negative or positive controls; therefore it is hard to analyze gels in Fig. 6. And 7. with certainty. Nonetheless, there are no significant differences between the lanes, especially around 79 kDa, so we can assume that overexpression did not occur in any of the samples.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-7-liquid-page-1.svg)

***Figure 7.** . First gel from SDS-PAGE screening for ScfL production after IPTG induction in liquid medium.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-8-liquid-page-2.svg)

***Figure 8.** . Second gel from SDS-PAGE screening for ScfL production after IPTG induction in liquid medium*

On the gels in figures 7. And 8. in lanes with PnbA-DocScaB and Dcx1-DocXylY we can see distinct thick bands confirming that overproduction occurred in the experiment conditions. We can also see a distinct band around 27.8 kDa in lane from a fluorescent colony, probably coming from sfGFP, which is absent in the rest of the lanes confirming that sfGFP gene was absent, or at least non-functional, in non-fluorescent colonies. There are, however, no bands around 79 kDa indicating ScfL production in any lane.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-9-bez-markera.svg)

***Figure 9.** . Colony-PCR of 50 colonies in groups of 5.*

Unfortunately, we have not added a stain to the marker, which is why it is not visible in figure 9. Nevertheless, we can see that product was created in every lane, so there were sequences complementary to the primers in at least one colony in every group. There is always only a single band in every lane and always on the same height, so even though we cannot check its length, it is likely that scaffoldin sequence is present in every group of colonies. In the lanes on the right, the negative controls were separated, and no band is visible, confirming that all the visible bands are the products of PCR reaction.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-10-colony-pcr.svg)

***Figure 10.** . Colony-PCR of colonies 11, 12, 13, 14 and 15.*

In every lane we can see a band between 500 bp and 400 bp marker bands. The expected PCR product length is 446 bp, therefore it shows that bacteria in colonies 11, 12, 13, 14 and 15 harbor the pJUMP23-1A vector with scaffoldin sequence. In the lane before the marker on the right, the negative control was separated and no band is visible, confirming that all the visible bands are the result of PCR reaction.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-11-sim-pcr-scfl.svg)

***Figure 11.** . Simulation of gel after PCR verification of ScfL ligation.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-12-pcr-scfl.svg)

***Figure 12.** . Simulation of gel after PCR verification of ScfL ligation.*

On the gel in figure 12. every control lane is empty, showing that all the bands come from the PCR reaction. In the lane with pJUMP\_1\_R reverse primer, there is a band that is only slightly higher than the simulation predicts (figure 11.). In the lane with RBS\_348\_R reverse primer, there is no band. This primer gave a very faint band in the PnbA-DocScaB vector analysis (figure 19.), so it might indicate that the primer is suboptimally designed. In the lane with Scaf\_R reverse primer, there is a band that is slightly higher than expected; the difference is non-negligible but might be a result of, for example, some secondary structure formation. In the lane with DT3\_274\_R reverse primer, there is a ladder. The thickest band is much lower than expected, but there is one band at the proper height. This primer had the highest melting temperature of 62.7 °C. If the sequence has low specificity, it might cause relatively strong non-complementary annealing. Together, it might result in most of the product sequences being unspecific. In the lane with T5E\_332\_R reverse primer, there is also a ladder, but the main band is only slightly higher than expected. The ladder might be caused by the primer’s low specificity to its sequences. In the lane with Term\_324\_R reverse primer, there is a band on the expected height. In the lane with Prom\_296\_F and pJUMP\_1\_R primer pair, there is also a band on the expected height.

In conclusion, although it is still possible that this vector was assembled properly, this gel has too many issues to prove it. This analysis should be repeated and the primers might need to be redesigned. Alternatively, sequencing could solve this ambiguity. Unfortunately, we did not have enough time for that.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **OBTAINING BACTERIA PRODUCING ENZYMES BY BLUNT-END CLONING**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

As mentioned before, we had troubles with Golden Gate assembly, so we ordered complete sequences of PnbA-DocScaB device ([BBa\_K4695210](http://parts.igem.org/Part%3ABBa_K4695210)), EstJ6-DocScaB device ([BBa\_K4695211](http://parts.igem.org/Part%3ABBa_K4695211)) and Dcx1-DocXylY device ([BBa\_K4695220](http://parts.igem.org/Part%3ABBa_K4695220)) from IDT. We wanted to test the expression of our enzymes from popular expression vectors, but our constructs were designed to have BsaI restriction sites for Golden Gate assembly. That limited our ability to clone it to multiple cloning sites of most vectors. Considering that, we decided to try blunt-end cloning to the pBAD24 vector, as the synthetized sequences had blunt ends. Next, *E. coli* strain Rosetta(DE3) cells were transformed with the obtained ligation mixture. The correctness of the cloning was assessed by restriction analysis.

## **EXECUTION**

1

#### Blunt-end cloning

[Blunt-end cloning protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/12.pdf)

2

#### Transformation of *E. coli* strain Rosetta(DE3)

---

Due to low efficiency, we did this step twice. We used 3 µl of ligation mixture.

[Transformation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/9.pdf)

3

#### Isolation of plasmid

[Plasmid isolation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/4.pdf)

4

#### Restriction analysis

---

We cut the plasmid with EcoRI, which should cut out gene parts, and EcoRV – the same enzyme used in cloning as a negative control, since this enzyme should recognize no sequences.

Restriction reaction mixtures were incubated at 37 °C for 2h for EcoRV, and 20 minutes for EcoRI. Both mixtures were kept at 80 °C for 20 minutes to stop the reaction.

Table 6. Restriction reaction mixture for EcoRI:

| Reagent | Volume added [µl] |
| --- | --- |
| Water | 18 |
| NEB rCutSmart™ Buffer | 2.5 |
| DNA | 4 |
| NEB EcoRI | 0.5 |
| Final volume | 25 |

Table 7. Restriction reaction mixture for EcoRV:

| Reagent | Volume added [µl] |
| --- | --- |
| Water | 17.5 |
| 10X Buffer R | 2.5 |
| DNA | 4 |
| Thermo Scientific? Eco32I (EcoRV) | 1 |
| Final volume | 25 |

Agarose gel electrophoresis was done in 1% agar 0,5 x TBE gel at 100 V. We used 5 µl of NEB Purple 1 kb+ ladder as marker and A&A 6x loading buffer mixed with GelRed to a final concentration of 0,6%.

## RESULTS

There were very few colonies after transformation with Dcx1-DocXylY and PnbA-DocScaB, and there were no colonies of EstJ6-DocScaB. Therefore, we did not do restriction analysis for vector expressing EstJ6-DocScaB.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-13-blunt-end.svg)

***Figure 13.** Gel after blunt-end cloning restriction analysis.*

Plasmid containing Dcx1-DocXylY sequence was expected to have 5603 bp. After cutting with EcoRI, there should be 3838 bp, 1366 bp and 333 bp fragments visible. Plasmid containing PnbA-DocScaB sequence was expected to have 6605 bp. After cutting with EcoRI, there should be 1177 bp and 5428 bp fragments. On the gel in figure 13. in the lanes with uncut plasmid, bands are much higher than expected, which might indicate that ligation occurred incorrectly. Perhaps, multiple linearized vectors have connected to each other. There are no 1366 bp and 1177 bp bands, which indicates that there were no accessible EcoRI restriction sites in the plasmids, which suggests that cloning did not go as expected.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **OBTAINING BACTERIA PRODUCING ENZYMES (VECTOR PUC)**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

Vector pUC ([BBa\_J435330](http://parts.igem.org/Part%3A)) was the first vector we had chosen as a potential expression vector for our devices, as it is a part of the *E. coli* expression kit collection in the iGEM Distribution Kit. We cloned sequences of each device encoding Dcx1-DocXylY, PnbA-DocScaB and EstJ6-DocScaB to the pUC vector using Golden Gate assembly. Then, we transformed *E. coli* strain BL21(DE3) with the obtained plasmid.

During proper Golden Gate assembly sticky ends of ligated DNA fragments should assemble into sequences recognized by SapI restriction enzyme. Therefore, we assessed the correctness of the ligation by restriction analysis with SapI. It should result in the formation of two bands on the agarose gel with the following lengths:

* Dcx1-DocXylY: 1764 bp and 1042 bp
* PnbA-DocScaB: 1764 bp and 2044 bp
* EstJ6-DocScaB: 1764 bp and 1417 bp

We used two negative controls, one was an undigested plasmid, and the other was treated with BsaI which should not recognize any sequence on the vector after proper assembly.

## **EXECUTION**

1

#### Golden Gate assembly

---

We assembled 3 vectors, each containing one of the following devices: Dcx1-DocXylY, PnbA-DocScaB and EstJ6-DocScaB.

Deviation from the protocol: we used the protocol for library preparation.

[Golden Gate protocol for 2 parts](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/8.pdf)

2

#### Transformation of *E. coli* strain BL21 (DE3)

---

We used 3 µl of ligation mixture.

[Transformation protocol for *E. coli* strain BL21 (DE3)](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/9.pdf)

3

#### Isolation of plasmid

[Plasmid isolation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/4.pdf)

4

#### Restriction analysis

---

We modified the protocol of restriction with SapI and BsaI in order to save on the enzymes. Reaction mixtures for both enzymes were filled according to table 8. The reaction was conducted at 37 °C for 20 minutes and the enzymes were inactivated at 80 °C for 20 minutes.

Table 8. Modified reaction mixture for BsaI and SapI.

| Reagent: | Volume: |
| --- | --- |
| Nuclease free water | 20 µl |
| NEB rCutSmart™ Buffer | 2.5 µl |
| DNA | 2 µl |
| Enzyme (BsaI or SapI) | 0.5 µl |
| Total volume | 25 µl |

Restriction analysis was conducted in the same agarose gel as the analysis of experiment 3. We used 5 µl of NEB Purple 1kb+ ladder as marker and A&A 6x loading buffer mixed with GelRed to a final concentration of 0,6%.

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-14-vect-d1-e1-e2.svg)

***Figure 14.** Restriction analysis of the vectors for overexpression of PnbA-DocScaB, Est6J-DocScaB and Dcx1-XylY with vector pUC backbone.*

On the gel in figure 14. we can see all the expected bands in lanes treated with SapI which indicates, that SapI restriction sites were successfully formed. Lanes with uncut plasmid and treated with BsaI look the same which indicates that BsaI restriction sites were successfully removed from the vector pUC. In figure 2. we can see that the unmodified vector pUC is cut by BsaI. We can conclude, that the assembly was carried out correctly.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **SCREENING OF PJUMP VECTORS**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

Originally we planned to conduct overexpression of our devices from vector pUC (BBa\_J435330), but we came to conclusion, that this high copy number plasmid can be suboptimal for overexpression, so we decided to try another vector. While looking for alternatives we found JUMP part collection in iGEM Distribution Kit, consisting of various plasmids and decided to test a few of them. We chose the plasmids listed in table 9. All of them had constitutively active sfGFP gene. We transformed them do DH5α and inoculated overnight liquid culture with transformants to see how intensely they would fluoresce.

Table 9. pJUMP plasmids we chose and transformed

| Our name system | Registry ID part | Number from the list | Plate number / Well number | Prefix and suffix | Length | Concentration (ng/ul) | *E. coli* strain the part was transformed into |
| --- | --- | --- | --- | --- | --- | --- | --- |
| pJUMP 23-1A | BBa\_J428347 | 273 | 2/E9 | AF | 3952 bp | 32 | DH5α |
| pJUMP 26-1A | BBa\_J428350 | 305 | 2/E17 | AF | 3163 bp | 26 | DH5α |
| pJUMP 28-1A | BBa\_J428353 | 129 | 1/A10 | AF | 3359 bp | 30 | DH5α |
| pJUMP 29-1A | BBa\_J428341 | 124 | 1/G8 | AF | 3815 bp | 36,5 | DH5α |

## **EXECUTION**

1

#### Transformation of *E. coli* strain DH5α with pJUMP vectors

[Transformation protocol for DH5α](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/9.pdf)

2

#### Inoculation of liquid bacterial culture and comparison of GFP fluorescence

---

5 ml of LB + 30 µg/ml kanamycin was inoculated with colony of *E. coli* transformed with each of the 4 vectors and left overnight with shaking. Culture was then observed under UV light of wavelength 365 nm.

3

#### Isolation of plasmid

[Plasmid isolation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/4.pdf)

4

#### Measurement of plasmid concentration

[Protocol for Nanodrop](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/7.pdf)

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-15-pjumps-fluor.jpg)

***Figure 15.** Fluorescence comparison between bacteria with different pJUMP vectors. From the left: pJUMP23-1a, pJUMP26-1A, pJUMP28-1A, pJUMP29-1A.*

The most intense fluorescence was observed in pJUMP28-1A and the least intense in pJUMP23-1A. Because sfGFP has been expressed constitutively and is a protein with relatively low requirements, we assumed, that fluorescence intensity in this case is correlated with plasmid copy number. We were looking for a low copy number vector, as we expected it to work better for overexpression. Therefore for future use we chose pJUMP23-1A.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **OBTAINING BACTERIA PRODUCING ENZYMES (PJUMP23-1A)**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

We had concluded that pJUMP23-1A would be the most promising available vector for the expression of our devices, therefore we cloned the devices encoding Dcx1-DocXylY, PnbA-DocScaB and EstJ6-DocScaB ordered from IDT into this plasmid. Then we transformed *E. coli* strain BL21(DE3) which is tailored for gene overexpression. Next, we isolated the plasmid and conducted fast verification by restriction analysis followed by more complex PCR verification. We performed 16 PCR reactions with the following primers pairs:

* fJUMP\_1\_F and pJUMP\_1\_R
* fJUMP\_1\_F and RBS\_348\_R
* fJUMP\_1\_F and Est\_1\_R
* fJUMP\_1\_F and DT4\_357\_R
* fJUMP\_1\_F and Ock\_2\_R
* fJUMP\_1\_F and T5E\_332\_R
* fJUMP\_1\_F and Term\_324\_R
* Prom\_296\_F and Est\_1\_R

Every pair was used in two reactions: one with template DNA and one without template DNA as a negative control. Sequences complementary to the primers are shown in figure 14. This way we wanted to verify if all the parts were assembled correctly.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-16-1-e1-map-png.png)

***Figure 16.** Scheme of pJUMP23-1A-pnbA-docscaB expression vector with primers used in this experiment shown in purple.*

## **EXECUTION**

1

#### Golden Gate cloning of ordered devices to pJUMP23-1A

---

We used the protocol for library preparation.

[Golden Gate assembly protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/8.pdf)

2

#### Transformation of *E. coli* strain BL21(DE3)

[Transformation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/9.pdf)

3

#### Plasmid isolation

[Plasmid isolation protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/4.pdf)

4

#### Verification of proper construct assembly with restriction analysis

---

We treated each of the 3 vectors and unmodified pJUMP23-1A with XbaI alone and with a combination of XbaI and BamHI. XbaI alone linearizes the unmodified pJUMP23-1A and cuts out short 113 bp parts from the same expression vectors containing our devices, which on the gel gives the same effect as linearization. XbaI and BamHI combination should have the same effect as sole XbaI on unmodified pJUMP23-1A, but cut out parts of a specific length from correctly cloned expression vectors:

* for pnbA-docscaB – 950 bp, 630 bp and 303 bp
* for estJ6-docscaB – 1310 bp
* for dcx1-docxylY – 881 bp

The rest of the vector should form a band of 3152 bp.

5

#### Verification of proper pJUMP23-1A-pnbA-docscaB construct assembly with PCR

---

16 PCR tubes were filled as shown in table 10. Primers were added in the volume of 1.25 µl each. Template DNA stands for isolated plasmid diluted in NF water in proportion 1:9. Reagents were mixed on ice and centrifuged briefly after mixing. Thermocycler was programmed according to the table 11. PCR products were separated in 1% 0.5xTBE agarose gel electrophoresis with the voltage of 100 V. We used 5 µl of NEB Purple 1kb+ ladder as a marker and A&A 6x loading buffer mixed with GelRed to a final concentration of 0,6%.

Table 10. PCR tubes content

| No. | Forward primer | Reverse primer | Template [µl] | Master Mix [µl] | NF water [µl] |
| --- | --- | --- | --- | --- | --- |
| 1 | fJUMP\_1\_F | pJUMP\_1\_R | 1 | 12.5 | 9 |
| 2 | fJUMP\_1\_F | pJUMP\_1\_R | 0 | 12.5 | 10 |
| 3 | fJUMP\_1\_F | RBS\_348\_R | 1 | 12.5 | 9 |
| 4 | fJUMP\_1\_F | RBS\_348\_R | 0 | 12.5 | 10 |
| 5 | fJUMP\_1\_F | Est\_1\_R | 1 | 12.5 | 9 |
| 6 | fJUMP\_1\_F | Est\_1\_R | 0 | 12.5 | 10 |
| 7 | fJUMP\_1\_F | DT4\_357\_R | 1 | 12.5 | 9 |
| 8 | fJUMP\_1\_F | DT4\_357\_R | 0 | 12.5 | 10 |
| 9 | fJUMP\_1\_F | Ock\_2\_R | 1 | 12.5 | 9 |
| 10 | fJUMP\_1\_F | Ock\_2\_R | 0 | 12.5 | 10 |
| 11 | fJUMP\_1\_F | T5E\_332\_R | 1 | 12.5 | 9 |
| 12 | fJUMP\_1\_F | T5E\_332\_R | 0 | 12.5 | 10 |
| 13 | fJUMP\_1\_F | Term\_324\_R | 1 | 12.5 | 9 |
| 14 | fJUMP\_1\_F | Term\_324\_R | 0 | 12.5 | 10 |
| 15 | Prom\_296\_F | Est\_1\_R | 1 | 12.5 | 9 |
| 16 | Prom\_296\_F | Est\_1\_R | 0 | 12.5 | 10 |

Table 11. PCR program

|  | Initial denaturation | Denaturation | Annealing | Elongation | Final elongation | Hold |
| --- | --- | --- | --- | --- | --- | --- |
| Temperature | 98°C | 98°C | 65°C | 72°C | 72°C | 10°C |
| Time [s] | 30 s | 10 s | 30 s | 80 s | 120 s | indefinite |
| Number of repeats | 1 | 30x |  |  | 1 | 1 |

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-17-pjump-rest.svg)

***Figure 17.** Verification of proper pJUMP23-1A vector constructs assembly with restriction analysis*

We can observe all of the expected bands, except for 303 bp in PnbA-DocScaB, which probably were too short to be visible or have been washed out of the gel. This suggests that all three vectors were assembled correctly.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-18-sim-pcr-e1.svg)

***Figure 18.** Simulation of gel with PCR analysis of pJUMP23-1A-pnbA-docscaB vector assembly in SnapGene*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-19-pcr-e1.svg)

***Figure 19.** 1% agarose gel with PCR analysis of pJUMP23-1A-pnbA-docscaB vector assembly*

All the expected bands appear on the gel at the correct height confirming, that the vector has been assembled correctly.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **COMPARISON OF PNBA-DOCSCAB, ESTJ6-DOCSCAB AND DCX1-DOCXYLY PROTEIN OVERPRODUCTION**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

We managed to obtain *E. coli* clones with vectors encoding PnbA-DocScaB, EstJ6-DocScaB and Dcx1-DocXylY with vector pUC (BBa\_J435330) or pJUMP23-1A (BBa\_J428347) backbones. We tried overproducing these proteins overnight at 16 °C on a small scale to see if there was any difference in the abundance of enzymes between vector pUC and pJUMP23-1A on the gel. After discovering that pJUMP23-1A gives better yield, we also tried more intense 3-hour overproduction at 37 °C with CaCl2 supplementation, since dockerin domains have binding pockets for calcium ions, determining their folding. Therefore we wanted to see if it could enhance the overproduction.

## **EXECUTION**

1

#### Overnight overexpression in pUC (BBa\_J435330) vector

---

We inoculated liquid culture in LB + 30 µg/ml kanamycin and left it overnight at 37 °C. The next day we refreshed the culture by adding 2 ml of the culture to 50 ml of LB + 30 µg/ml kanamycin and at OD 600 =0.8 we induced overproduction by adding IPTG to a final concentration of 1 mM. Then we left the culture overnight in 16 °C. We took samples of 1 ml right before induction and 0.4 ml the next day after overexpression. The samples were then centrifuged and the pellet was lysed in Laemmli buffer by heating to 80 °C for 20 minutes and separated in SDS-PAGE. For the first 15 minutes voltage was 120 V, and then it was increased to 180 V for the rest of the separation.

[SDS-PAGE protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/13.pdf)

2

#### Overnight overexpression in pJUMP23-1A (BBa\_J428347) vector

---

It was done the same way as with vector pUC:

We inoculated liquid culture in LB + 30 µg/ml kanamycin and left it overnight at 37 °C. The next day we refreshed the culture by adding 2 ml of the culture to 50 ml of LB + 30 µg/ml kanamycin and after OD 600 reached 0.8 we induced overproduction by adding IPTG to a final concentration of 1 mM. Then we left the culture overnight at 16 °C. We took samples of 1 ml right before induction and 0.4 ml the next day after overexpression. The samples were then centrifuged and the pellet was lysed in Laemmli buffer by heating to 80 °C for 20 minutes and separated in SDS-PAGE. For the first 15 minutes voltage was 120 V, and then it was increased to 180 V for the rest of the separation.

[SDS-PAGE protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/13.pdf)

3

#### Short time overexpression in pJUMP23-1A (BBa\_J428347) vector with CaCl2

---

We inoculated liquid culture in LB and left it overnight at 37 °C. The next day we refreshed the culture by adding 2 ml of the culture to 50 ml of LB + 30 µg/ml kanamycin and at OD 600 =0.5 we induced overproduction by adding IPTG to a final concentration of 1 mM and additionally CaCl2 to the concentration of 5 mM. Then we left the culture for 3.5 hours at 37 °C. We took samples of 1 ml right before induction and 0.4 ml after the overproduction. The samples were then centrifuged and the pellet was lysed in Laemmli buffer by heating to 80 °C for 20 minutes and separated in SDS-PAGE. For the first 15 minutes voltage was 120 V, and then it was increased to 180 V for the rest of the separation.

[SDS-PAGE protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/13.pdf)

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-20-overprod-in-vect.svg)

***Figure 20.** SDS-PAGE after overnight overexpression from vector pUC.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-21-pj23-overprod-overnight.svg)

***Figure 21.** SDS-PAGE after overnight overexpression from pJUMP23-1A.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-22-overprod-mieszny.svg)

***Figure 22.** SDS-PAGE after 3.5 hour overexpression from pJUMP23-1A.*

Our fusion proteins are expected to have following molar mass:

* PnbA-DocScaB – 64,6 kDa
* EstJ6-DocScaB – 42,0 kDa
* Dcx1-DocXylY – 28,4 kDa

Comparing gels from overnight overexpression we can see that expected bands are much thicker in samples from pJUMP23-1A than those from vector pUC. In EstJ6-DocScaB case there isn’t even a visible band in samples from vector pUC. Also, bands heights seem much more accurate in pJUMP23-1A which might indicate that enzymes in cells with vector pUC didn’t have correct conformation. It might be caused by high copy number of vector pUC, which is often disadvantageous for protein overexpression. Meanwhile pJUMP23-1A has medium copy number. Coming to these conclusions, we decided to use vector with pJUMP23-1A backbone for protein production.

Comparing gels from overnight and 3.5-hour overexpression we can see that induced bands are much thicker in samples from overnight overexpression. Again, there is no visible band for EstJ6-DocScaB in samples from 3.5-hour overexpression. The OD600 after overproduction reached 0.7, which was quite low. Perhaps we added too much of CaCl2. Therefore, we decided to carry out overnight overexpression for production of these enzymes.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **OBTAINING PURIFIED PNBA-DOCSCAB AND DCX1-DOCXYLY**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

After obtaining bacteria with functional expression vector encoding PnbA-DocScaB and Dcx1-DocXylY we needed to induce overexpression of those sequences and harvest proteins on a big scale. After that, we set to purify the protein using immobilized metal affinity chromatography (IMAC). We used Ni-NTA resin, since our proteins were His-Tagged at the C-terminal end.

## **EXECUTION**

1

#### Big scale overproduction

---

We inoculated 6 liters of LB medium with *E. coli* strain BL21(DE3) containing two vectors: pJUMP23-1A-pnbA-docscaB and pJUMP23-1A-dcx1-docxylY (3 liters each). IPTG induction was done when OD600 reached 2.5. Ideally it should be performed at values ranging from 0.5-0.8. Unfortunately, the scale-up of the process changed the dynamic of bacterial growth and we missed the optimal spot. Cultures were incubated at 16 °C overnight with shaking 160 RPM. After that, cells were harvested by centrifugation at 4500 RCF and the product – bacterial paste containing desired proteins – was frozen.

2

#### Bacterial lysis

---

Half of the bacterial paste with PnbA-DocScaB and half of the paste with Dcx1-DocXylY was resuspended in lysis buffer and lysed by sonication. Then both lysates were centrifuged at 75 000 RCF to separate soluble and insoluble fractions. All the samples were checked for the presence of the proteins of interest on SDS-PAGE.

[SDS-PAGE protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/13.pdf)

3

#### Affinity chromatography on Ni-NTA resin

---

Supernatants with both proteins were loaded on the Ni-NTA resin and washed with 50 mM imidazole buffer. Then the 50-500 mM gradient of imidazole was applied. SDS-PAGE gel was performed with fractions collected from both samples’ purification. Samples were chosen based on the UV absorbance signal acquired during purification. After visualization on gel, fractions 15-24 from the PnbA-DocScaB gradient were collected.

4

#### Plasmid concentration measurement

---

10 fractions containing PnbA-DocScaB were mixed (total volume: 20 ml) and dialyzed to the final buffer overnight (1:100 sample:buffer ratio). On the next day purified protein solution was portioned and frozen. Densitometry was performed with a BSA calibration curve, and the protein concentration was established using ImageLab software.

[Protein purification protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/14.pdf)

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-23-big-scale-overprod.svg)

***Figure 23.** SDS-PAGE gel containing samples before and after induction of a large scale (3 liters each) overproduction of PnbA-DocScaB and Dcx1-DocXylY proteins. Every sample is doubled on the gel.*

Induction of both cultures was successful, as additional bands appear on the gel after adding IPTG and incubating overnight. The thickness of the bands is not as big as in the test overproduction, most probably because of the late induction. The PnbA-DocScaB band is on the expected height (64,6 kDa), but the band expected to be Dcx1-DocXylY (28,6 kDa) is much lower, around 20 kDa. This indicates, that the protein was not expressed properly. Lack of around 7 kDa can correspond do the molecular weight of the dockerin domain, which is 7 kDa as well.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-24-page-from-wirowanie.svg)

***Figure 24.** SDS-PAGE gel containing samples of lysates, supernatants and pellets of the bacteria harboring overproduced proteins PnbA-DocScaB and Dcx1-DocXylY. This was done to establish which fraction should be loaded onto the Ni-NTA resin. The gel used was flawed, but it is still possible to gather data from the results.*

Results indicate, that most of the PnbA-DocScaB is located in the supernatant and most of the overproduced protein from the pJUMP23-1A-dcx1-docxylY is located in the pellet. We opted to purify the supernatant with PnbA-DocScaB and due to the lack of time needed for optimization and purifying proteins from pellet we also decided to try to load the potential Dcx1-DocXylY supernatant onto the Ni-NTA resin, as there is a small band of overproduction in the supernatant sample.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-25-oczyszczanie-frakcje.svg)

***Figure 25.** SDS-PAGE gel containing samples from different stages of Ni-NTA purification of PnbA-DocScaB.*

It can be observed, that the PnbA-DocScaB band (64,6 kDa) visible in the load sample is much less abundant in the flow-through fraction, which indicates binding of the PnbA-DocScaB to the resin. Wash (50 mM imidazole) eluted most of the non-specifically bound proteins. Imidazole gradient (50-500 mM) proved to be effective in eluting the desired protein. At a concentration of around 150 mM of imidazole, a thick band with height corresponding to the overproduction band in the first path started to appear on the gel. The purity and the concentration of eluted samples are decent.

![](https://static.igem.wiki/teams/4695/wiki/website/general-notebook/figure-26-oczyszczanie-d1.svg)

***Figure 26.** SDS-PAGE gel containing samples from different stages of Ni-NTA purification of Dcx1-DocXylY.*

It can be observed, that most of the overproduced protein is still visible in the flow-through fraction. This, in addition to the lack of any specific bands at the expected kDA in next steps, shows the absence of desired protein in the supernatant load. In 10th and 12th fractions two bands at around 70 kDa height can be observed being eluted by higher concentrations of imidazole. Those are undesired proteins, which bind more specifically to the Ni-NTA resin, possibly due to higher histidine content.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **OBTAINING NATIVE PNBA BY TEV CLEAVAGE**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

Since we incorporated TEV protease recognition site between the enzymatic domain PnbA and dockerin domain DocScaB we wanted to obtain the native form of PnbA. It was supposed to be used in the enzymatic analysis to compare its activity with the fusion protein. Unfortunately we didn’t have enough time to conduct such analysis. After the TEV protease cleavage we tried to separate the native enzyme from cleaved dockerin domain on the Ni-NTA resin taking advantage of the fact that the His-tag remained linked to the dockerin.

## **EXECUTION**

1

#### Enzymatic reaction

---

TEV protease was borrowed from the resources of the Laboratory of Protein Biochemistry from our faculty. It was not a commercially available protein, and we used ratios according to the Laboratory’s protocols. 3 ml of the purified protein solution was mixed with 200 µl of TEV protease solution. This reaction mixture was left overnight at 400 RPM shaking at 16 °C.

2

#### Purification

---

On the next day mixture of PnbA-DocScaB and TEV protease was loaded onto a small amount of Ni-NTA resin in batch in order to separate PnbA and DocScaB domains. Elution was done with the same buffer, as during the protein purification (500 mM imidazole). The flow-through and elution fractions were collected. Samples of load, flow-through and elution were collected along the way to visualize the process on a SDS-PAGE gel.

3

#### Assessment of cleavage and purification

---

Fractions collected in the previous step were applied to SDS-PAGE.

[SDS-PAGE protocol](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/13.pdf)

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-27-tev.svg)

***Figure 27.** SDS-PAGE gel with fractions obtained from enzymatic digestion of PnbA-DocScaB cut with TEV protease.*

In the path with purified PnbA-DocScaB we can see a thick band of the protein with little contaminations. In the path with the post-reaction mixture (PnbA-DocScaB + TEV protease) we can see a thick band of PnbA and a band of cleaved DocScaB, both on the expected height, proving that protein was successfully cleaved. We can also see a distinct band at the same height as uncut enzyme, meaning that the cleavage was not complete and in the mixture remained significant amounts of whole PnbA-DocScaB. In the path with Ni-NTA flow-through we can see all of the bands that we could observe in the post-reaction mixture, meaning that the purification was not very efficient. In the path with Ni-NTA elution we can once again see all 3 bands of PnbA-DocScaB, PnbA and DocScaB, but thinner, especially the PnbA. It means that even though the purification was not satisfactory some of the cleaved dockerin domain was removed. Surprisingly PnbA was also present in the elution, meaning that, even though His-tag should have been removed together with dockerin domain, native form of PnbA seems to still have some affinity to the Ni-NTA resin.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **ANALYSIS OF THE THERMOSTABILITY OF PNBA-DOCSCAB ON TYCHO (NANOTEMPER)**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

Analysis of the purified protein on SDS-PAGE is conducted in denaturing conditions, so no information about protein folding and activity is obtained. After purification of PnbA-DocScaB, we wanted to characterize the acquired enzyme in terms of its stability and tertiary structure.

## EXECUTION

Usage of Tycho is very simple. According to the protocol, that guides you when using the system, in order to measure our sample we simply left it to reach the room temperature, and then transferred a fraction of the purified protein solution to the dedicated capillary and inserted the capillary into the device. The analysis was done, data were collected and analyzed.

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-28-tycho-raw.svg)

***Figure 28.** Graph showing the relation of temperature with the ratio of intrinsic fluorescence at 350 nm and 330 nm, which directly translates to the folding state of measured protein.*

Tycho allows to see how the rise of the temperature affects the tertiary structure of proteins in the sample, by measuring the intrinsic fluorescence of tryptophan and tyrosine residues. The higher the ratio, the more residues are emitting light and the more unfolded the protein is. Results of this particular experiment show a big unfolding event occurring at around 54 °C and a smaller one at around 63 °C. Event at 54 °C might correspond to the unfolding of the enzymatic PnbA domain, and the 63 °C can be associated to the unfolding of the smaller dockerin domain. This shows, that the purified PnbA-DocScaB protein indeed has a functional tertiary structure and loses stability at >50 °C.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **PLOTTING A CALIBRATION CURVE OF STANDARDS ON HPLC**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

In order to measure the concentrations of substrates and products of enzyme reactions in High-Performance Liquid Chromatography (HPLC) we needed to plot a calibration curve. We worked on two different chromatographs, therefore we made the calibration curve twice.

## **EXECUTION**

1

#### Preparing stock solutions of standards

---

We used dibutyl phthalate (DBP), monobutyl phthalate (MBP), butyl benzoate (BB), phthalic acid (PA) and benzoic acid (BA). DBP, MBP an BB were prepared in 1 M concentration in acetonitrile (ACN), PA and BA were prepared in 0.1 M because of limited solubility in ACN. PA was prepared in 50% ACN in 10 mM PBS (pH = 7.4), because it didn't dissolve completely in pure ACN. Stock solutions were prepared in 1 ml, except for MBP, which was made in 0.1 ml, because we acquired it only in limited amount.

Table 12. Properties of HPLC standards stock solutions.

| Standard | Pure substance state of matter | Molar mass [g/mol] | Amount of standard added | Stock solution volume [ml] | Stock solution concentration [mol/dm3] |
| --- | --- | --- | --- | --- | --- |
| dibutyl phthalate (DBP) | liquid | 278.34 | 266.9 µl | 1 | 1 |
| monobutyl phthalate (MBP) | powder | 222.24 | 22.2 mg | 0.1 | 1 |
| butyl benzoate (BB) | liquid | 178.23 | 176.47 µl | 1 | 1 |
| phthalic acid (PA) | powder | 166.13 | 16.61 mg | 1 | 0.1 |
| benzoic acid (BA) | powder | 122.12 | 12.21 mg | 1 | 0.1 |

2

#### Mixing calibration curve samples

---

We made samples containing all 5 standards. At first we prepared 100 mM solutions of each standard, then mixed them in 10 mM solution. Next, two solutions were prepared: 2500 µM and 2000 µM. So far all the dilutions were made in ACN. Then 500 µM and 400 µM dilutions were prepared by addition of 40 µl of 2500 µM or 2000 µM solution accordingly and filling to 200 µl with 10 mM PBS (pH = 7,4). Then rest of the concentrations used for plotting the calibration curve were prepared in 20% ACN in PBS. 500 µM solution was further diluted 1:1 2 times to prepare 250 µM and 125 µM dilutions. Similarly 400 µM solution was diluted in 1:1 4 times to prepare 200 µM, 100 µM, 50 M and 25 µM. For the first chromatograph we used all 8 concentrations. For the second one we used only 500 µM, 400 µM, 250 µM, 125 µM, 25 µM, as we decided it is sufficient and wanted to save on the HPLC mobile phase.

3

#### Running the analysis

---

We transferred 50 µl of each concentration to autosampler bottles. Then 30 µl was loaded on the Phenomenex Jupiter 5um C18 300A (250x4.6mm). 1 hour gradient of mobile phase (0% to 100% buffer B, buffer A – dH2O with 0,1% trifluoroacetic acid – TFA, buffer B – 80% ACN in dH2O with 0,1% TFA) at 40 °C was applied to separate the samples.

## RESULTS

We needed to estimate low concentrations near the end of the calibration curve concentration range. Therefore, considering that no peak on the chromatogram was observed with no substance added, we decided to set the calibration curve intercept at (0,0).

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-29-curve-1.svg)

***Figure 29.** Calibration curves for the first chromatograph with intercept set at (0,0).*

Table 13. Properties of calibration curves

| Compound | R² | equation | Wavelength [nm] |
| --- | --- | --- | --- |
| BA | 0,998 | y = 17,834x | 205 |
| PA | 0,9951 | y = 25,42x | 205 |
| BB | 0,9578 | y = 1,3646x | 225 |
| MBP | 0,9913 | y = 22,186x | 205 |
| DBP | 0,9807 | y = 9,0715x | 225 |

Calibration curves plotted for PA. MBP and BA on the first chromatograph have coefficient of determination (R2) >0.99, giving quite a good fit. Calibration curves plotted for DBP and BB on the other hand have quite low R2 therefore values calculated using them would not be very reliable.

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-30-curve-2.svg)

***Figure 30.** Calibration curves for the second chromatograph with intercept set at (0,0).*

Table 14. Properties of calibration curves

| Compound | R² | equation | Wavelength [nm] |
| --- | --- | --- | --- |
| BA | 0,9928 | y = 6,0545x | 205 |
| PA | 0,9906 | y = 20,236x | 205 |
| BB | 0,9957 | y = 1,526x | 205 |
| MBP | 0,99 | y = 21,752x | 205 |
| DBP | 0,9903 | y = 6,0077x | 205 |

All calibration curves plotted on the second chromatograph have R2 equal or higher than 0.99 giving a decent fit. Surprisingly, peak areas for BB are higher than on the first chromatograph, even though they were measured at wavelength of 205 nm, at which BB has lower molar absorption coefficient than at 225 nm used in the first chromatograph.

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-gora.svg)

## **ANALYSIS OF PNBA-DOCSCAB ACTIVITY AGAINST DBP AND BB**

![](https://static.igem.wiki/teams/4695/wiki/website/parts/naglowek-dol.svg)

## PURPOSE

After purifying PnbA-DocScaB we wanted to test its activity. The analyses were done in 20% ACN in 10 mM PBS (pH = 7.4) to increase substrate solubility. We used dibutyl phthalate (DBP) and butyl benzoate (BB) as substrates. This experiment was carried out in triplicate for both DBP and BB. Enzymatic reaction was stopped by centrifugation on a membrane with 10 kDa weight cut off. This also separates proteins from the rest of the sample, preventing them from being loaded onto the HPLC column. Concentrations of the compounds were calculated using the second callibration curve (figure 30.)

## **EXECUTION**

1

#### Setting up enzymatic reaction

---

We mixed 5ml of reaction mixture consisting of 20% ACN in PBS, 400 µM of dibutyl phthalate (DBP) or butyl benzoate (BB) and 223 nM (14.41 µg/ml) of purified PnbA-DocScaB. Reaction was set up in room temperature. Simultaneously the control sample was prepared with 50 µl of 10% glycerol in PBS instead of 50 µl of the enzyme. It is the same buffer in which the enzyme was dissolved.

Enzymes were added last and right after that mixtures were vortexed and aliquots of 500 µl were sampled.

Then the mixtures were kept for 30 minutes at 37 °C with shaking. After this time aliquots of 500 µl were taken from each sample again.

2

#### Sample preparation

---

Aliquots were directly applied to an Amicon® Ultra 0.5 ml and centrifuged at 10 000 RCF for 10 minutes.

3

#### Sample analysis on HPLC

---

We transferred 50 µl of filtrate from each sample to an autosampler bottle. Then 30 µl were loaded on the Phenomenex Jupiter 5um C18 300A (250x4.6mm). 1 hour gradient of mobile phase (0% to 100% buffer B, buffer A – dH2O with 0,1% trifluoroacetic acid – TFA, buffer B – 80% ACN in dH2O with 0,1% TFA) at 40 °C was applied to separate the samples.

[Protocol for Nanodrop](https://static.igem.wiki/teams/4695/wiki/website/experiments/protocols/7.pdf)

## RESULTS

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-31-dbp.svg)

***Figure 31.** Mean DBP concentration in samples after centrifugation on Amicon, measured at 205 nm.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-32-mbp.svg)

***Figure 32.** Mean MBP concentration in samples after centrifugation on Amicon, measured at 205 nm.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-33-bb-tycie-svg.svg)

***Figure 33.** Mean BB concentration in samples after centrifugation on Amicon, measured at 205 nm.*

![](https://static.igem.wiki/teams/4695/wiki/website/experiments/figure-34-ba-tycie-svg.svg)

***Figure 34.** Mean BA concentration in samples after centrifugation on Amicon, measured at 205 nm.*

We can see that the concentration of substrate in control sample is around ten times lower than 400 µM in DBP in time 0 and almost four times lower after 30 minutes. In the case of BB it even dropped below the calibration curve concentration range. It indicates that this method requires redesigning. Amicons might be the potential cause of the concentration decrease. It requires investigation if these compounds interact with the filter. This experiments should be repeated with different method of reaction stopping and enzyme removal. We can see that in the control sample there is no MBP but in the samples with PnbA-DocScaB there has been a clear signal from MBP on the chromatogram. Similarly in the experiment with BB as a substrate in the control sample there have been minimal peak from BA, but there have been a significant signal from BA in the sample with the enzyme. It should be kept in mind that we cannot say with confidence what was the concentration of the product in the sample, since substrate concentration measurements in control samples turned out to be unreliable. Nonetheless this data proves that PnbA-DocScaB catalyzes reactions of ester bond hydrolysis in both DBP and BB. Though there was no signal from PA, therefore we cannot say if this enzyme catalyzes hydrolysis of MBP in the experiment conditions.

We can see that with sampling time equal 0 minutes product was already created. It indicates that reaction stopping by centrifugation on Amicon was too slow to observe the change in reagents concentration in time. If we were to repeat this experiment we would try stopping the reaction with acid as it was dome by Xu et al. (Xu Youqiang, 2021). Reaction mixture setting and sample preparation have been done in room temperature, therefore we can see that enzyme was functional in this temperature. Concentration of substrate significantly changes after 30 minutes in both control and research sample. Moreover it increases for DBP and decreases for BB. These unexpected changes in substrate concentration might be connected to the temperature change during incubation, since DBP and BB did not form homogenous mixture in 20% ACN in PBS, so temperature rise might have changed the distribution of the substrates between phases.

It is important to point out that after 30 minutes of incubation white flakes precipitated from the reaction mixture. It was probably denatured enzyme. It shows that PnbA-DocScaB has rather low stability in 20% ACN in PBS in these temperature conditions. Fast denaturation of the enzyme also gives plausible explanation for lack of product concentration rise after 30 minutes of incubation.

In conclusion, although this experiment failed to give data about reaction kinetics and enzyme activity it still confirms that PnbA-DocScaB catalyzes ester bond hydrolysis in MBP and BB in room temperature.

u Youqiang (2021) ‘An efficient phthalate ester-degrading *Bacillus subtilis*: Degradation kinetics, metabolic pathway, and catalytic mechanism of the key enzyme’, Environmental Pollution, 273, p. 116461. Available at: https://doi.org/10.1016/j.envpol.2021.116461.

{% endblock %}

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{% block title %}Results{% endblock %}
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## **INTRODUCTION**

We are proud of our results obtained during our past ten months in iGEM 2023 competition lab work. We were fortunate to have learned more then we could expect. We managed to perform multiple experiments (Golden Gate assemblies, Restriction analyses, PCRs, transformations, overproductions, purifications and HPLC runs) and each of them brung us closer to the reaching the finished project. All of the experiments performed and all of our results are meticulously described in [the experiments subpage](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) of our 2023 iGEM Wiki.

On this page we want to briefly show our most important conclusions, highlighting positive and negative outcomes of our efforts. We also present our future plans, as the project is far from being over and we want to continue developing our ideas and gathering data to confirm our theories.

## **IMPORTANT HIGHLIGHTS**

### WHAT WE ARE PROUD OF

* It was our first time working with the iGEM 2023 DNA Distribution Kit Plate. We learned how to design functioning devices with its contents and how to use the deposited parts in wet lab.
* We aqcuired a library of *E. coli* clones harbouring parts from iGEM DNA Distribution Plate Kit that we needed to assembly our constructs.
* We learned how to use the RFC1000 system to construct desired parts and devices.
* We learned and tested different cloning methods (Golden Gate assembly, Blunt End ligation) which allowed us to obtain desired constructs.
* We performed dozens of restriction analyses and some PCR analyses to verify the correctness of our cloning experiments.
* We tried different conditions for the overproduction of PnbA-DocScaB and chose the optimal one for the large scale overproduction.
* We overproduced and purified the PnbA-DocScaB fusion protein.
* We determined the stability of PnbA-DocScaB.
* We proved that PnbA-DocScaB catalyses ester bond hydrolysis in dibutyl phthalate (DBP) and butyl benzoate (BB).

### WHAT WE REGRET

* We failed to achieve protein expression from constructs assembled using separate parts via Golden Gate assembly. We optimised the method for scaffoldin construct and right now the PCR results are ambigous - we can confirm presence of the coding sequence but we are still unsure of the proper assembly. Unfortunately, still no protein expression was observed.
* Despite promising results about obtaining EstJ6-DocScaB, Dcx1-DocXylY and ScfL-His we did not manage to fully confirm the correct assembly of those constructs. Fortunately, we have the tools to achieve that in the future.
* We did not have time to fully characterize the PnbA-DocScaB enzymatic activity.
* We managed to overproduce and describe only 1 out of 7 designed constructs. Although we are unhappy because of this result, we are also hungry for more and cannot wait to continue our project after the competition.

## **OUR FUTURE PLANS**

**Although we have worked hard and produced plenty of data, we did not manage to complete all of our ambitious plans. Here, we have described the experiments we want to conduct in the future, as we did not have enough time to complete them during our iGEM competition participation. They are listed in order that we will probably follow, given a chance to continue our endeavors:**

* We want to repeat the PnbA-DocScaB activity tests using different method, that would allow as to reliably measure the changes in the reagents concentration and also gather information about reaction kinetics. Currently we suspect that stopping the reaction with a strong acid and then extracting the reagents with a organic solvent, such as ethyl acetate.
* We want to test PnbA-DocScaB activity against as the only available substrate MBP, as we tested this activity only indirectly, in the analysis where it was the product of DBP hydrolysis.
* We want to compare the activity of PnbA-DocScaB and native form PnbA obtained by TEV protease cleavage.
* We want to test PnbA-DocScaB activity in different conditions of pH, temperature, ionic strength and acetonitrile content.
* Prof. Dr. Jörn Kalinowski Head of Technology Platform Genomics, Center for Biotechnology from Bielefeld University have offered us the service of nanopore sequencing, so we want to sequence pJUMP23-1A-pnbA-docscaB vector and pJUMP23-1A-scfL-docscaB vector to finally confirm that they were assembield correctly.
* We want to purify Dcx1-DocXylY from the pellet after cell lysis as it seems to be insoluble.
* We want to test the activity of Dcx1-DocXylY against monobutyl phthalate (MBP) and phthalic acid PA.
* If sequencing indicates that our pJUMP23-1A-scfL-docscaB vector was not assembled correctly, we will modify the sequence of ScfL-His amplicon with PCR to add appropriate enzyme restriction sites in primer overhangs that would allow us to clone this sequence to pCOLADuet-1. We want to test if the assembly and overexpression of our device would be easier in videly recognised expression vector might. Appropriate primers for this procedure have already been synthetised.
* An alternative way of obtaining functional expression vector for scfL gene would be synthetising the complete scfL device with all the regulatory sequences and clone it to pJUMP23-1A vector as one insert.
* We want to obtain the other decarboxylases: Dcx2-DocXylY, Dcx3-DocXylY, Dcx4-DocXylY and together with Dcx-DocXylY test if they have the desired decarboxylase activity against MBP and PA. If more than one if them catalyse this reaction we will compare their activity to find the best one.
* If we find functional decarboxylase we will obtain their native forms by TEV protease cleavage and compare their activity to their conterparts with dockerin domains.
* We want to test the affinity of ScfL to the cellulose by packing fine cellulose beads into a column and load a sample containing ScfL onto the column. We would compare the initial sample and the flow through in SDS-PAGE. We could try eluting the protein from the column by applying a gradient of increasing ionic strength to test the stability of the immobilisation.
* We want to test the if the enzymes bind to the ScfL. We could do that by first immobolising the ScfL on the cellulose bed in the column in the column and then loading the samples with enzymes onto the column. Comparison of input and output samples on SDS-PAGE would tell us if the binding occured. The results would be compared with the control with no ScfL on the column.
* The interaction of enzymes containing dockerin domain with the ScfL could also be tested by microscale thermoforesis it.
* We want to test the activity of enzymes immobilised to the ScfL and then compare the results with the free enzymes.
* We want to test the activity of our protein complexes in the filter by filling the filter with a cellulose medium and filling the filter with water containing DBP and then measuring the changes of DBP and its degradation products concentration on HPLC.
* We want to test the efficiency of the filter by filling it with cellulose medium with enzymes immobilised and pass a certain volume of water with known concentration of DBP and then measure the concentration of DBP and its degradation products in the effluent.

**These ideas constitute a path that we envision before us, leading us to our goal - water safe from phthalates pollution, both for the people and for the wild life. We hope that our effort will make a contribution in solving the global problem of endocrine disrupting chemicals. We are aware that down the road there will be many unexpected complications to resolve and difficult decisions to make. Nevertheless we are determinated to press forward and excited to explore all the vast possibilites to make our region a safe place to live and play a part in ensuring the sustainability of our civilization.**

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