Team:TUDelft/Notebook/Bacteriophages
Notebook
Bacteriophages
Week 24
Week 25
Week 26
Week 27
Week 28
6/7
Maartje & Iris:
Plasmid G322-pUC57-OriLR-deGFP was kindly provided by the Danelon lab. To confirm the expression of GFP and to obtain a glycerol stock, G322-pUC57-OriLR-deGFP was transformed into chemical competent E.coli BL21(DE3) cells (Invitrogen), following the protocol for heat shock transformation, with minor adjustments. 10 ng DNA was added to 25µl of cells and incubated on ice for 15 minutes, followed by 30 seconds heat shock at 42ºC. 250µL of pre-heated LB medium was added to recover the cells. The cells were plated on LB-agar plates with ampicillin and incubated overnight.
To check for contamination, a control was done using 1 µL of water instead of DNA.
7/7
Maartje & Iris:
After incubation, the control did not show any colonies. The plates containing the transformed cells did show colonies. One colony was picked and grown overnight in 10 ml LB-amp to obtain a fresh culture for a glycerol stock.
Week 29
14/7
Maartje & Iris:
BL21(DE3)::pKD46 To prepare electrocompetent cells for both BRED and regular electroporation reactions, BL21(DE3) and BL21(DE3)::pKD46 was grown overnight at 30C in respectively 10ml LB medium and 10 ml LB-amp medium.
1 ml of each overnight culture was put to grow in 100 ml LB or 100 ml LB-amp at 30C until early log phage (OD600~0.4-0.6). 0.1% L-arabinose was added to BL21(DE3)::pKD46 for 30 minutes. 2 ml of T7 phage was added and incubated for 15 minutes, resulting in an MOI of 2. Both batches of cells were made electrocompetent following the protocol for [electrocompetent cell preparation.]
Week 30
Week 31
31/7
Javier
Construction of the dsDNA fragment for phage engineering
- Resuspension of primers for phage insert production (GFP 1.1 Rv and Fw, 0.6 Rv and Fw, 4.6 Rv and Fw)
- KOD Xtreme amplification to produce the GFP inserts for the phage [KOD Xtreme], Annealing 58ºC 30'', 68ºC 1'.
Week 32
2/8
Maartje & Iris:
To obtain a fresh culture of BL21(DE3), BL21(DE3) was put to grow overnight in 10ml LB medium.
3/8
Maartje & Iris:
Bacteriophage recombineering of electroporated DNA was performed following the protocol for [BRED], electroporating 200ng and 500ng of each dsDNA substrate. After recovery the suspension was mixed with 100µl fres BL21(DE3) culture and 3 ml 0.6% top agar and incubated overnight at 37C.
4/8
Maartje & Iris:
No single plaques were observed, the control with only a bacterial lawn did show full growth of bacteria, control with wild-type T7 did not show any growth of the bacteria and the sample obtained after BRED showed some bacterial growth.
14/7
Maartje & Iris:
1L Phage buffer (1xPBS, 1mM MgCl2, 1mM MgSO4) was prepared following the protocol for phage buffer preparation.
Week 33
10/8
Maartje & Iris:
To obtain a fresh culture for more electrocompetent cell preparation, BL21(DE3)::pKD46 was put to grow overnight at 37C instead of the recommended 30C.
11/8
Maartje & Iris:
Electrocompetent cells were prepared containing pKD46 and T7 phage at an MOI of 1 and 2 following the protocol for electrocompetent cell preparation for [BRED].
12/8
Maartje & Iris:
BRED was performed using approximately 250ng dsDNA as substrate. To investigate the effect of MOI on the efficiency of BRED electrocompetent cells were used containing a T7 MOI of 1 and 2. After plating of the suspension, the plates were checked every 2 hours, but no bacterial growth was observerd, indicating a high concentration of phages.
Week 34
18/8
Maartje
To obtain a fresh culture for a glycerol stock, BL21(DE3)::pKD46 was put to grow overnight in 10ml LB-amp medium.
To optimize BRED, electroporation was performed with only electrocompetent BL21(DE3)::pKD46 with T7. The different conditions were different MOI (1 and 2) and different voltages (1.8 and 2.5 kV). Before electroporation, the electrocompettent cells were two times diluted in 10% glucerol. Before plating, the suspensions were 10 times serial diluted in phage buffer until 10^8.
19/8
Maartje
Single plaques were observed in plates with the 10^8 times diluted phage suspension.
A [glycerol stock] of BL21(DE3)::pKD46 was obtained following the protocol for glycerol stock.
The presence of pKD46 was checked doing a miniprep of the fresh culture of BL21(DE3)::pKD46.
19/8
Maartje
A gradient PCR was performed to obtain the optimal annealing temperature for the PCR with the T7 flanking primers (M1-001 - M1-006) using GoTaq polymerase following the PCR protocol for [GoTaq]. The annealing temperature was put at 44-58C, 1µL of T7 wild type phage (3*10^10 pfu/ml) was used as template DNA and the number of cycles was 30.
Week 35
27/8
Maartje
BRED was executed following [BRED] with minor adjustments. After recovery in SOC medium, the suspension was ten fold serial diluted in phage buffer. Each dilution was added to 300µL fresh BL21(DE3) cells and 3 ml 6% topagar and poured over a bottom agar layer and incubater overnight at 37C.
To check the expression of GFP on the plasmid, BL21(DE3)::GFP was streaked on LB-amp plates and incubated overnight at 37ºC.
28/8
Maartje
GFP check: all plates show fluorescence.
BRED: there were no fluorescent plaques shown. Of each reaction 20 plaques were picked and dissolved in 100µl phage buffer and incubated at RT for 1 hour. The gradient PCR of 20/8 was checked on 1.5% agarose gel. For each primer 56.9 is the optimal annealing temperature. PCR with 1 µl of the plaque solutions was performed using the flanking primers, following the protocol for [GoTaq].
Week 36
1/9
Maartje
The results of the flanking plaque PCR were loaded on a 1.5% agarosegel. Al the lengths of the DNA fragments were the same as the control, so in the plaques screened, no engineered phage was present.
To confirm the presence of pKD46 in the electrocompetent cells used for BRED, a restriction reaction of the miniprep solution of 19/8 was executed using BamHI-HF, following this protocol.
2/9
Maartje
Since the results of the PCR showed no amplification for phage plaques 5, 9, 13, 17 and 19 from the engineering with the dsDNA matching gene 0.6, these samples were checked for the presence of phages using a spot assay. 100µl of BL21(DE3) overnight culture was added to 3 ml top agar and poured on a bottom agar layer and incubated for approximately 10 minutes. 3 µl of each plaque solution was spotted on the pate and the plate was incubated overnight at 37C.
3/9
Maartje
Plaque check
Plaques were shown in the plates obtained from the spot assay, so the PCR using flanking primers M1-003 and M1-004 of plaque 5, 9, 13, 17, 19 was repeated, again the results did not show any amplification of the phage DNA.
4/9
Maartje
Phage infectivity assay
To be able to compare the infectivity of the engineered phages and the wildtype phages, [phage infectivity assays] were done. [Phage] titer was determined.
Week 37
8/9
Maartje
To check the presence of the GFP gene in plaques 5, 9, 13, 17 of gene insertion 0.6, PCR was done using primerpair M1-003, M1-048 and M1-009, M1-010 and 35 cycles.
9/9
Maartje
The PCR products were loaded on 1.0% [agarosegel].. A population PCR of the filtered BRED samples after recovery in SOC medium was done using for each reaction each flanking primer pair and the forward flanking primer with primer M1-048.
10/9
Maartje
The PCR samples were loaded on a 1.0% agarosegel. The PCR reaction using forward flanking primers and M1-048 showed in each reaction sample amplification, indicating that in each filtered BRED sample there are engineered phages present.
Week 38
14/9
Maartje
Because the results of 10/9 did show the presence of the GFP gene in the engineered phages, but only with the forward flanking primers and the primer M1-048, PCR reaction was done using these primers on each phage plaque sample. These samples were loaded on a 1.5% agarosegel showing no amplification of phage DNA, indicating that there are no engineered phages in the plaque samples.
15/9
Maartje
To obtain the engineered phage, the fitness of the wildtype phage was decreased by the reduction of oxygen. For this 300µl of fresh culture of BL21(DE3) was added to 45 ml of LB medium in a 50 ml tube and incubated for 1.5h at 150 rpm. Each phage filtrate (filtered samples of recovered cells after BRED) was added to the tube and the mixture was incubated for 3h ate 37C at 150 rpm. Each solution was filtered with a 45µm filter and the phage titer was determined by a spot assay. The titer of each phage filtrate was 10^8 pfu/ml.
Phage activity assay
The phage activity assay of the wildtype phage was done again, this time in duplo following this protocol. Due to technical deficiencies, this time the bacteria and phages were not incubated at 180 rpm, but only shaken briefly every five minutes.
Week 39
22/9
Maartje
Reduction fitness wt T7
The samples of 15/9 obtained after the oxygen reduction experiment were diluted until 10^5 and added to 300µl cells to 3ml 7% top-agar and incubated for 4 hours at 37C. 30 plaques were picked and dissolved in phage buffer
22/9
Maartje
Phage characterisation
To determine the pfu of the samples taken for phage characterization on 15/9, a spot assay was performed.
Reduction fitness wt T7
A PCR was performed on the samples obtained after the oxygen reduction experiment using the flanking primers M1-001 - M1-006 and a primer annealing to GFP following the PCR protocol for[GoTaq].
24/9
Maartje
To obtain a plasmid carrying sgRNA targeting the wildtype phage, two PCRs were performed on pKDsgRNA-p15. The forward primers M1-049, M1-052, M1-053 and reverse primer BetaR were used for the first reactions, and the forward primer PkdseqF with the reverse primers M1-050, M1-052, M1-054 for the second reactions. The protocol for Q5 polymerase was followed. The PCR products were checkedn on a 1% agarosegel and subsequently a Gibson Assembly was done with the two obtained PCR fragments. The products were transformed following the protocol for heat shock transformation into dh5alpha. The cells were recovered at 30C and plated on LB-spec.
Week 40
30/9
Gabriela
Phage experiment plaque size over time in collaboration with Vienna.
300ul of overnight BL21(DE3) + 100ul T7 phage (10^3, 10^2, 10^1 pfu/ml) in 3ml TOP agar, on LB agar plates. With this experiment we want to compare the plaque size and the appearance of the plaque. This way our premade phages are compared to Vienna's encoded phage that they expressed.
Results: Too many plaques; there is overlap. Possibly due to wrong dilution.
Do it again tomorrow with more dilutions
Week 41
6/10
Maartje
A co-transformation of Cas9-CR4 together with pKDsgRNA-0.6, 1.1 and 4.3 into BL21(DE3) was done following the protocol for electroporation. Cells were recovered at 30C and plated on LB-Spec/Cam plates.
A colony of BL21::Cas9-CR4 was picked and put to grow overnight in LB-Cam, to prepare a fresh culture for electrocompetent cell preparation.
A infection dose assay was done with T7 wildtype phage. In stead of a starters OD600 of 0.4, the OD600 was ~0.5.
7/10
Maartje
Electrocompetent BL21::Cas9-CR4 cells were prepared following the protocol for electrocompetent cell preparation. A transformation to these cells was done with pKDsgRNA-0.6, -1.1 and -4.3. Cells were recovered on LB-Spec/Cam and LB-Spec as a control.
8/10
Maartje
Positive colonies with Cas9-CR4 and pKDsgRNA-0.6 and -4.3 of the transformation of 6/9/2020 were picked and grown overnight and streaked on LB-Spec plates and LB-Cam separately to check for both plasmids.
9/10
Maartje
All the overnight cultures grew and the results of the colonies streaked on both antibiotics indicate that both plasmids were present in the cultures.
Week 42
12/10
Maartje
The entire PCR product of the screening of the plaques was loaded on a 1% agarose gel. Results showed that one plaque of the 4.3 engineered phage contained the insert.
13/10
Maartje
This plaque was serial diluted and plated on e.coli expressing Cas9 and sgRNA4.3. 24 secondary plaques were screened with internal primers following the protocol for [GoTaq]. Polymerase.
14/10
Maartje
The PCR products were loaded on a 1% agarose gel. Results show bands around 300bp.
15/10
Maartje
Flanking PCR was performed with primers M1-005 and M1-006 on plaque number 9, 11 and 15 of the day before following the protocol for GoTaq Polymerase. The PCR product was loaded on a 1% agarose gel.
