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Starting culture
Colonies were growing on both the positive control and the LB + amp. plates with the transformed cells!
Cloning of the pMK1 vector with a restriction ligation using enzymes SacI and NdeI to incorporate the fourfold resilin repeat (RE4).
Protocol 3 Digest with Restriction Enzymes was used for the double digestion of pMK1.
Restriction mix contained:
Component | Volume |
DNA | 1 µg |
10X rCutSmart Buffer | 5 µL |
NdeI | 0.5 µL |
SacI-HF | 0.5 µL |
Nuclease-free Water | ad 50 µL |
Components | Volume |
---|---|
10X T4 DNA Ligase Buffer | 2 µL |
Vector (digested pMK1) | 0.56 µL |
Insert (digested RE4) | 0.6 (1:5); 0.32 (1:3); 0.13(1:1) |
T4 DNA Ligase | 1 µL |
Nuclease-free water | ad 20 µL |
Primer | FW_pMK1_RE4_G->C |
RW_pMK1_RE4_G->C | |
Template DNA | pMK1_RE4_1:5_2 |
pMK1_RE4_1:5_3 | |
pMK1_RE4_1:3_2 | |
pMK1_RE4_1:3_4 |
Templates used:
Step | Temperature [°C] | Time [s] | Cycles |
---|---|---|---|
annealing | 69 | 30 | 30 |
elongation | 72 | >100 | 30 |
Results of the competence test: Transformation of pMK1 into TOP10 was successful, but BL21 bacteria did not grow
Step | Temperature [°C] | Time [s] | Cycles |
---|---|---|---|
annealing | 50 | 30 | 30 |
elongation | 72 | >120 | 30 |
Transformation of pMK1 into TOP10 and BL21* competent cells following protocol 2 Transformation.
Changes:
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.
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.
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 |
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 |
Step | Temperature [°C] | Time |
---|---|---|
Annealing | 54 | 30 s |
Extension | 72 | 80 s |
Final extension | 72 | 3 min |
DpnI Digestion:
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 |
Sample | Vector [ng] | Ratio [insert:vector] | Volume [µL] |
---|---|---|---|
1 | 129.7 | 1:2 | 10 |
2 | 37 | 1:4 | 10 |
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 |
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.
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 |
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.
The overnight cultures grew well and were sent in for sequencing. They came all back negative for the insert.
For the isolation protocol 7.2 was followed.
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).
The pUC-HasA PCR from the 13.09.2024 was repeated.
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.
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
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 |
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
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).
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