01.07.2024

Testing competent cells with pMK1

Transformation of pMK1 into TOP10 and BL21* competent cells following protocol 2 Transformation.
Changes:

• After adding the pre-warmed SOC medium the cells were incubated for 60 min on the shaker at 37 °C and 300 rpm
• 100 µL of the bacteria suspension were plated on the LB-Agar-Amp. Plates
• As a negative control the competent cells were incubated in 100 µl water and plated on the LB-Agar-Amp

03.07.2024

Transformation of DH5α with pUC_HasA

• Wet the dry pUC_HasA plasmid DNA
• Transformation of pUC_HasA into DH5α following protocol 2 Transformation.

Changes:

• After adding just 200 µL pre-warmed SOC medium the cells were incubated for 60 min on the shaker at 37 °C and 300 rpm
• 250 µL were plated on LB-Agar-Amp. Plates

04.07.2024

Overnight culture of transformed DH5α with pUC_HasA for expression of hyaluronic acid synthase

• Two colonies (pUC_HasA_1 and pUC_HasA_2) from the transformation of pUC_HasA in DH5α from 03.07.24 were picked and transferred in two tubes with 3 mL of LB-medium.
• Additionally 3 µL of ampicillin (100 mg/mL stock solution) were added for a 1:1000 dilution.
• Finally the cell suspensions were incubated over night at 37 °C, 150 rpm on a shaker

05.07.2024

Miniprep of pUC_HasA for expression of hyaluronic acid synthase

A miniprep was carried out with the over night cultures from pUC_HasA_1 and pUC_HasA_2 from 04.07.24 following the protocol 7.1 Plasmid Mini-Prep - NEB.

16.07.2024

Transformation of pUC_HasA in TOP10 for expression of hyaluronic acid synthase

• Transformation of pUC_HasA_1 and pUC_HasA_2, purified with a miniprep on 04.07.24, into competent TOP10 bacteria following the protocol 2 Transformation..
• A total of 100 µL of bacteria suspension with pUC_HasA were plated on the LB-Agar-Amp. Plates.
• As a negative control water was added to the competent cells.

17.07.2024

Overnight culture of transformed TOP10 with pUC_HasA for expression of hyaluronic acid synthase

• Three colonies from the tranformation of pUC_HasA_2 in TOP10 performed on 17.07.24 were picked and placed in three tubes with 3 mL of LB-medium each.
• Additionally 3 µL of ampicillin (100 mg/mL stock solution) were added to achieve a 1:1000 dilution.
• Finally the cell suspensions were incubated over night at 37 °C, 150 rpm on a shaker.

18.07.2024

Induction of hyaluronic acid synthase expression with arabinose

• For the initiation of the hyaluronic acid synthase expression 50 mL LB-Medium with 50 µL of ampicilin (100 mg/mL stock solution) were added to the 2 mL of overnight cultures 2 and 3 from 17.07.2024.
• The cell cultures were incubated at 37 °C, 130 rpm on the shaker until they reached an OD600 of 0.6.
• As a control 1 mL of the cell culture from clone 2 and 3 were taken at an OD600 of 0,6 and stored at 4 °C until further analysis.
• To start the synthetisation, 500 µL of arabinose (10 g/L stock solution; final concentration = 1 µg/mL) were added to the culture of clone 2 and 1000 µL of arabinose (final concentration = 2 µg/mL) were added to the culture of clone 3.
• The cell cultures of clones 2 and 3 were then incubated for 20 h at 37 °C, 150 rpm on a shaker.

19.07.2024

Analysis of the hyaluronic acid synthase expression with an SDS-PAGE

After 20 h expression of Hyaluronic acid through induction with arabinose the OD600 of the cultures from clones 2 and 3 were measured.

Culture (Arabinose conc.)	OD600 (20 h after induction)
Clone 2 (1 µg/mL)	2,750
Clone 3 (2 µg/mL)	N/A

300 µL of Clone 2 and 3 were diluted with 700 µL LB medium to reach an OD600 of 1. Even tho clone 3 was not measurable undiluted, it had an OD600 of 1 after adding the LB medium.

To analyze the hyaluronic acid expression an SDS-Page with an 12% gel was performed following the protocol 9 SDS-PAGE.

To prepare the samples under reducing conditions for the SDS-PAGE 50 µL of clones 2 and 3, both pre and post induction, were mixed with 50 µL of reducing sample buffer and a total of 15 µL were then loaded onto the gel. As a ladder the PageRuler Unstained Protein Ladder (Thermo Scientific, Cat. no.: 26614) was used.

After the SDS-PAGE the gel was stained with Coomassie for 1 h and destained with ddH2O over night.

Expected bands: 52 kDa (HasA)




Results:
We were able to see bands at a height of about 52 kDa, which is consistent with the molecular weight of the hyaluronic synthase.
In the next steps, we choose to prove and observe the hyaluronic acid expression with an HasA-eGFP fusion protein.

24.07.2024

Cloning of a HasA-eGFP fusion protein for the detection of HasA Expression

To insert the eGFP+Linker behind the HasA gene a SLiCE reaction will be used. For the SLiCE the two fragments eGFP+Linker and pUC_HasA are linearized with a PCR reaction containing homologue overhangs.

The PCRs were performed following the protocol 5.1 PCR - Q5 Mastermix from NEB with following templates, primers and annealing temperatures:

Reaction	Template	Primer	Annealing Temperature [°C]	Elongation [s]
HasA_SLiCE_1	pUC_HasA	pUC_IDT_hasA_fw_SLICE & pUC_IDT_hasA_rev_SLICE	57.1	185
HasA_SLiCE_2	pUC_HasA	pUC_IDT_hasA_fw_SLICE & pUC_IDT_hasA_rev_SLICE	60.9	185
eGFP_SLiCE_1	eGFP_Linker	GFP_fw_SLICE & GFP_rev_SLICE	55	40
eGFP_SLiCE_2	eGFP_Linker	GFP_fw_SLICE & GFP_rev_SLICE	56.4	40
eGFP_SLiCE_3	eGFP_Linker	GFP_fw_SLICE & GFP_rev_SLICE	57.7	40
eGFP_SLiCE_4	eGFP_Linker	GFP_fw_SLICE & GFP_rev_SLICE	59.1	40

To determine if the PCRs were successful, we ran agarose gels produced after protocol 8 Agarose gel.

expected bands:

5250 bp (HasA_SLiCE)

762 bp (eGFP_SLiCE)

Results: With bands being between 700-800 bp and the size of our desired product being 762 bp, eGFP_SLiCE PCR can be considered successful as suggested by band size

Problem: there were no bands at the expected 5250 bp. Retry with lower annealing temperature.

03.08.2024

PCR to amplify HasA_SLiCE fragment

• PCR according to protocol 5.1 PCR - Q5 Mastermix from NEB with pUC_IDT_hasA_fw_SLICE and pUC_IDT_hasA_rev_SLICE as primers, 55 °C annealing temperature and 3:30 min elongation time.
• 2.52 µL of pUC_HasA template were used.

To determine if the PCR was successful, we ran an agarose gel produced after protocol 8 Agarose gel.

expected bands: 5250 bp (HasA_SLiCE)


Result: With expected bands at 5250 bp, this result suggests that the PCR was successful and our next step is the SLiCE recombination with both the HasA_SLiCE and the eGFP_SLiCE.

08.08.2024

Gel Clean up of HasA_SLiCE and eGFP_SLiCE

• To retrieve the PCR product of the successful HasA_SLiCE and eGFP_SLiCE PCRs from the gel, we did a Gel clean up according to protocol 6.1 Gel clean up - Promega gel and PCR clean up.
• The PCR products can now be used in the SLiCE recombination to obtain the pUC_HasA_eGFP fragment

12.08.2024

SLiCE recombination with HasA_SLiCE & eGFP_SLiCE

• Recombination of purified pUC_HasA (see 03.08.24) and eGFP_SLiCE (see 24.07.24) with SLiCE according to protocol 10.1. Homologous Recombination - SLiCE.
• 8 samples were prepared, of which 4 were prepared in duplicates, differing in incubation times:

Sample	Vector [ng]	Ratio [insert to vector]	Volume [µL]	Incubation time
1	48	1:7	20	1x 1 h, 1x over night
2	67	1:3	20	1x 1 h, 1x over night
3	96	1:7	40	1x 1 h, 1x over night
4	134	1:3	40	1x 1 h, 1x over night

• Transformation of pUC_HasA-eGFP_SLiCE into TOP10 according to protocol 2 Transformation after 1h and over night incubation, respectively.

14.08.2024

Colony PCR of TOP10 - pUC_HasA_eGFP_SLiCE

• Preparation of the PCR according to protocol 5.3 PCR - DreamTaq Mastermix with the following primers, annealing and extension temperatures:



Step	Temperature [°C]	Time
Annealing	54	30 s
Extension	72	80 s
Final extension	72	3 min



• Primers: Rev_ColPCR_HasA_EGFP and FW_ColPCR_HasA_EGFP
• Template: Picked 6 pUC_HasA_eGFP_SLiCE colonies from two plates after 1h incubation as well as 6 from two plates after incubation over night.
• Positive control with 0.5µL of pUC_HasA in a 25µL sample

15.08.2024

Agarose Gel for the Colony PCR of TOP10 - pUC_HasA_eGFP_SLiCE

• Colony PCR samples from the day prior (14.08.2024) were run on a gel, prepared as described in protocol 8 Agarose gel.

16.08.2024

DpnI digestion and Repetition of the SLiCE recombination from 12.08.2024 with HasA_SLiCE & eGFP_SLiCE

DpnI Digestion:

• The two pUC_HasA_SLiCE fragments and four eGFP_SLiCE fragments were pooled, respectively.
• Concentrations were measured:
• pUC_HasA_SLiCE: 28.6 ng/μL
• eGFP_SLiCE: 20.7 ng/µL
• Pooled samples were digested with DpnI according to protocol 11 DpnI digestion.
• Incubation with DpnI was reduced to 1 h

SLiCE recombination:

• Recombination was preformed similar to the previous attempt of 12.08.2024 with the pooled and digested fragments pUC_HasA and eGFP_SLiCE according to protocol 10.1. Homologous Recombination - SLiCE.
• Insert to vector ratios were adjusted, total volume was reduced and 2 samples were prepared, of which each was prepared in duplicates, differing in incubation times:



Sample	Vector [ng]	Ratio [insert:vector]	Volume [µL]	Incubation time
1	85.8 [ng]	1:1	10	1 h / over night
2	28.6 [ng]	1:10	10	1 h / over night

17.08.2024

Transformation of pUC_HasA-eGFP_SLiCE-1 and -2 in TOP10

• Transformation of new Plasmids into TOP10 according to 2 Transformation
• DNA used, optimized for SLiCE to 2µL of the recombined product
• four were Plates prepared:
• two for pUC_HasA-eGFP_SLiCE-1, incubated for 1h and over night
• two for pUC_HasA-eGFP_SLiCE-2, incubated for 1h and over night

18.08.2024

Homolog recombination of the HasA and EGFP fragment using NEBuilder

• New approach for the recombination of pUC_HasA_SLiCE and eGFP_SLiCE according to protocol 10.2 Homologous Recombination - NEB-builder
• Total volume was reduced to 10 µL

Sample	Vector [ng]	Ratio [insert:vector]	Volume [µL]
1	129.7	1:2	10
2	37	1:4	10

26.08.2024

Transformation with the recombination products from 18.08.2024

• Transformation of pUC_HasA_eGFP_HR-1 and -2
• Transformation according to protocol 2 Transformation
• Transformed into TOP10
• 3µL recombination product was used

28.08.2024

Colony PCR of pUC_HasA_eGFP_HR

• 6 colonies were picked from pUC_HasA_eGFP_HR-1 and pUC_HasA_eGFP_HR-2, resulting in a total of 12 samples
• Colony PCR was performed as described in protocol 5.3 PCR - DreamTaq Mastermix
• Primers: Rev_ColPCR_HasA_EGFP and FW_ColPCR_HasA_EGFP
• Positive control: was added consisting of
• 12.5 μl DreamTaq Green PCR Master Mix (2x)
• 1.25 μl of each primer (pUC_IDT_hasA_fw_SLICE, Rev_ColPCR_HasA_EGFP)
• 0.5 μl pUC_HasA_eGFP
• 25 μl nuclease free water

All samples were negative!

04.09.2024

Colony PCR of the transformed SLiCE pUC_HasA_eGFP into Top10 bacteria

to test the colonies from the NEBuilder homolog recombination with pUC_HasA-  and eGFP-SLiCE-Fragments a Colony PCR was performed. 18 colonies were tested. 9 colonies were picked from the plate with a 1:2 vector: insert ratio. The other 9 colonies were picked from the plate with a 1:4 vector: insert ratio.
The Colony PCR Mastermix was done with 100 μl DreamTaq Green PCR Master Mix (2x), 10 μl of each primer Rev_ColPCR_HasA_eGFP and FW_ColPCR_HasA_eGFP and 80 μl nuclease-free water. The colonies were picked with a toothpick or a pipette tip and added into 10 μl PCR Mastermix. 12 colonies were tested. 6 colonies from the plates where the Top10 cells were transformed with the pUC_HasA-eGFP_SLiCE extract that was incubated for 1 hour and 6 from the plates where the extract was incubated overnight. A positive control was added consisting of 12.5 μl DreamTaq Green PCR Master Mix (2x), 1.25 μl of each primer (pUC_IDT_hasA_fw_SLICE, Rev_ColPCR_HasA_eGFP), 0.5 μl pUC_HasA_eGFP as Template and 25 μl nuclease free water.

The PCR was done as follows:

Step	Temperature, °C	Time	Number of cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	30
Annealing	54	30 s	30
Extension	72	1:20 min	30
Final Extension	72	3 min	1
Stop	10	infinite	1

The PCR was checked on a 1 % agarose gel (protocol 8).

07.09.2024

Second try to Transform the Homolog recombination of the HasA and eGFP fragment using NEBuilder

Transformation was done according to protocol 2 with 2 μl SLiCE-product (1:2 vector:insert ratio and 1:4 vector:insert ratio) and positive control and 1 μl negative control.

08.09.2024

Colony PCR after Transformation

The Colony PCR Mastermix was done with 50 μl DreamTaq Green PCR Master Mix (2x), 5 μl of each primer Rev_ColPCR_HasA_eGFP and FW_ColPCR_HasA_eGFP and 40 μl nuclease-free water. The colonies were picked with a toothpick or a pipette tip and added into 10 μl PCR Mastermix. 12 colonies were tested. 6 colonies from the plates where the Top10 cells were transformed with the pUC_HasA-eGFP_SLiCE extract that was incubated for 1 hour and 6 from the plates where the extract was incubated overnight. A positive control was added consisting of 12.5 μl DreamTaq Green PCR Master Mix (2x), 1.25 μl of each primer (pUC_IDT_hasA_fw_SLICE, Rev_ColPCR_HasA_eGFP), 0.5 μl pUC_HasA_eGFP as Template and 25 μl nuclease free water.

The PCR was done as follows:

Step	Temperature, °C	Time	Number of cycles
Initial denaturation	95	3 min	1
Denaturation	95	30 s	30
Annealing	54	30 s	30
Extension	72	1:20 min	30
Final Extension	72	3 min	1
Stop	10	infinite	1

From each plate 5 colonies were picked which resulted in 10 PCR tubes plus the positive control.
1: Colony of the 1:2 ratio plate
2: Colony of the 1:2 ratio plate
3: Colony of the 1:2 ratio plate
4: Colony of the 1:2 ratio plate
5: Colony of the 1:2 ratio plate
6: Colony of the 1:4 ratio plate
7: Colony of the 1:4 ratio plate
8: Colony of the 1:4 ratio plate
9: Colony of the 1:4 ratio plate
10: Colony of the 1:4 ratio plate

The PCR was performed with 60 °C annealing temperature and 2:20 elongation time for 30 cycles.

09.09.2024

Overnight culture

An overnight culture was prepared from the following samples: 5, 6, 7, 8, 9, 10. These showed bands at the right length and therefore should be checked via sequencing. The picked colonies were added to LB-Medium and put on the shaker at 37 °C overnight.

10.09.2024

Sequencing

The overnight cultures grew well and were sent in for sequencing. They came all back negative for the insert.

12.09.2024

Isolate the pUC-HasA by performing a miniprep

For the isolation protocol 7.2 was followed.

13.09.2024

Add overhangs at the puC-HasA for the homolog recombination

PCR according to protocol 5.1 with 30 cycles, annealing temperature 98 °C, and elongation for 3:20 min. Primers pUC_IDT_HasA_rev and pUC_IDT_HasA_fw were used.
Do a Dpn1 digestion to get rid of the template without overhangs and clean it up.
DpnI digest following protocol 3. 25 μl of PCR-product and 1 μl DpnI (NEB) were added to the reaction. Incubation for 2 hours at 37 °C, followed by a heat inactivation for 20 min at 80 °C. After that, a PCR purification was carried out according to protocol 6.1. The cleaned PCR product was analyzed on a 1 % agarose gel (protocol 8, figure 1.). There was no plasmid, so the HasA-PCR was repeated on the same day (figure2).

17.09.2024

pUC-HasA PCR

The pUC-HasA PCR from the 13.09.2024 was repeated.

18.09.2024

pUC-HasA and eGFP-SLiCE-PCR

pUC-HasA PCR according to protocol 5.2, with pUC_IDT_HasA_rev and pUC_IDT_HasA_fw for primers, 55 °C annealing temperature and 3:20 min elongation time. eGFP_SLiCE-PCR was also performed according to protocol 5.2 with GFP_fw_SLiCE and GFP_rev_SLiCE as primers. The elongation time was 1:20 min and the annealing temperature was 67 °C.

19.09.2024

DpnI digest and control via agarose gel

DpnI digest according to protocol 3 with 40 µl template, incubation for 2 hours at 37 °C, and heat inactivation for 20 min at 80 °C. The digest was put onto a 1 % agarose gel (according to protocol 8).

Gel:
1: Ladder 10 µl
2: HasA 2 (13.09.) undigested 9 µl
3: HasA  3.1 (18.09) 20 µl
4: HasA  3.2 (18.09) 20 µl
5: HasA  3.13(18.09) 20 µl
6: Ladder 10 µl
7: HasA  4.1 (18.09) 20 µl
8: HasA  4.2 (18.09) 20 µl
9: HasA  4.3 (18.09) 20 µl
10: Ladder 10 µl

After that a Gel purification was performed according to protocol 6.2, and the concentrations were measured.

Sample	Conc.	280/260	260/230
HasA CU1	16.5	1.73	0.12
HasA CU3	9.7	1.61	0.05
HasA CU4	11.2	1.53	0.13
HasA CU5	14	1.59	0.14
HasA CU6	18.2	1.66	0.15
HasA CU7	10.7	1.64	0.09

20.09.2024

NEBuilder Recombination

A 1 % agarose gel (according to protocol 8) was loaded with the eGFP-SLiCE-PCR product from the 18.09.

1: Ladder 1 kb
2: eGFP 1
3: eGFP 2

There were no bands (figure 1), so we started another eGFP-SLiCE-PCR according to protocol 5.2  but and used 69 ° degrees as a annealing temperature and 1:30 for elongation.
The we loaded yet another 1 % agarose gel (according to protocol 8) and filled the pockets with the new eGFP-PCR product just like above.
This time the PCR did work at least for eGFP 1 (figure 2).

23.09.2024

Amplification of the pUC_HasA_SLiCE Fragments (second try)

For the recombination of pUC_HasA, the fragment pUC_HasA will be amplified with primers containing the homolog regions in the overhang.

pUC_hasA-PCR (Protocol 5.2)
annealing temp 55°C
elongation: 3:20min

pUC_hasA from the overnight colony from 07.09.2024 diluted to 1ng/µl as template and we followed the protocol 5.2 with the following primers 10 µM pUC_HasA_fw_SLICE and 10 µM pUC_Has_rev_SLICE.
The PCR product was analysed on a 1% agarose gel (following protocol 8).

Afterwards, the Fragments were isolated from the Gel (protocol 6.2), adding a wash step with 750µL 70% ethanol right before the elution. Additionally, the ethanol was evaporated for 6min before the elution step. This extra step  was only done with pUC_HasA1 and pMK1_1.

pUC_HasA1: 53.6ng/ µL; 260/280 1.77; 260/230 1.25
pUC_HasA2: 64.8ng/µL; 260/280 1.72; 260/230 0.13
pMK1_1: 16.4ng/µL; 260/280 1.47; 260/230 0.75
pMK1_2: 31ng/µL; 260/280 1.42; 260/230 0.15

As one can see, the extra step resulted in a way better 260/230 ratio. So we decided to keep that step for any other Gel Clean Ups.

26.09.2024

GEL of eGFP from 27.09.24 + Isolation of good Bands

Making a 1% Agarose Gels with atotal Volume of 75 µL (2 µL of Midori Green pro Gel)
Loading the samples: 20 µL of each sample + 4 µL Loading dye
Ladder: 7 µL

Gel:
1. Ladder
2. eGFP 1 from 20.09
3. eGFP 2 from 20.09

Run the Gels at 120 V

cut out the band at eGFP 1

Clean up of eGFP 1
According to protocol 6.2 with the additional ethanol wash step before the elution
Weight of the gel pieces and amount of QB buffer
| eGFP | 229,6mg | 688, 8 µL |

Nanodrop measurment:
eGFP
19.9 ng/µL
260/280 1.87
260/230 0.21
(Dilution to 1 ng/µL   1 µL DNA + 19 µL water)

27.09.2024

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29.09.2024

Homologue recombination of pUC_HasA and eGFP

• Homologous recombination of puC_HasA and eGFP following the NEB-builder

30.09.2024

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01.10.2024

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