Team:TUDelft/Notebook/BPUL
Notebook
BPUL laccase and Decarboxylase
Week 24
Week 25
Week 26
Week 27
Week 28
7/7
Javier
Laccase
- 10ul of NF water were used to resuspend the BPUL laccase pasmid from iGEM2019 plates (plasmid: pSBIC3).
- 2ul used to transform 50ul of DH5-alpha cells following [DH5a transformation].
8/7
Javier
Laccase
- Colonies successfully grew o/n.
- 5 x 10ml cultures inoculated with 1 colony each. [10ml culture], o/n culture
- Control (-) with 10ml LB inoculated with no Ab.
9/7
Javier
Laccase
- None of the colonies inoculated grew o/n.
- Repeat 5 x 10ml cultures with 1 colony each [10ml culture], o/n culture.
- Control (-) with 10ml LB inoculated with no Ab.
10/7
Javier
Laccase
- Cultures did not grow, including control (-).
13/7
Javier
Laccase
- Transformation in DH5a [Transf. DH5a] with 2ul of DNA from (07/07/2020).
- control (-) with LB used in step 6.
Week 29
13/7
Alicia
Laccase
BPUL laccase transformation: BPUL laccase transformation
14/7
Alicia
BPUL laccase transformation: The transformation was not successful, as no colonies were obtained in the plates from 13-07-2020.
Javier
Laccase
- No growth in any of the petry dishes.
- Transformation in BL21 [Transf BL21], 2ul of DNA from (07/07/2020).
17/7
Javier
Laccase
- Transformation in DH5a [Transf. DH5a]
-
Plates
- LB-Chm (50ul of trasformants)
- LB-Chm (pellet)
- LB-SOC: control (-)
- LB-Chm: 5ul cells no plasmid, control (-)
- LB- no Ab: 5ul cells no plasmid, control (+)
18/7
Javier
Laccase
No transformants grew. No growth in negative controls, growth in positive control.
Week 30
22/7
Alicia
Transformation of the bsdBCD decarboxylase plasmid: The commercially cloned plasmid containing bsdBCD genes was delivered as dry powder (1999 ng). It was resuspended in 50 ul of Nuclease-Free H2O and incubated 5 min at 42ºC to facilitate DNA resuspension. 50 µl of BL21 (DE3) Competent E. coli (NEB) were transformed with 2 ul of DNA following the protocol for heat shock transformation. The plates were incubated overnight at 37ºC.
23/7
Alicia
Colonies were observed in the transformated plates from 22/07/2020.
Colonies were observed in the transformated plates from 22/07/2020.
24/7
Alicia & Javier
BPUL electroporation transformation: 4 µl of DNA were transformed into 20 µl of self-made BL21 (DE3) electrocompetent E. coli cells following the protocol for electroporation transformation. The plates were incubated in the dark at room temperature for the whole weekend.
Week 31
28/7
Alicia
Cry7Ca1 expression and bsdBCD decarboxylase expression
29/7
Javier
Plasmidic DNA isolation of pTWIST_Cry7Ca1 and pTWIST_bsdBCD: Preparation of 10 ml of cultures of LB media inoculated either with E.coli BL21 (DE3) pTWIST_Cry7Ca1 and E.coli BL21 (DE3) pTWIST_bsdBCD , with 10 µl of Ampicillin 100 mg/ml (1000X). The cultures were grown overnight at 37ºC and 200 rpm.
30/7
Javier
Plasmidic DNA isolation of pTWIST_Cry7Ca1 and pTWIST_bsdBCD.
Minipreps from the 10ml culture to ensure good purification of plasmid.
Week 32
Week 33
Week 34
Week 35
Week 36
23/9
Javier
SDS-PAGE electrophoresis for bsdBCD decarboxylase overexpression: Frozen pellets were unfrozen and resuspended in 100 µl of Nuclease-Free H2O. Samples to load in the gel were prepared by mixing 10 µl of the resuspensions with 10 µl of Sample Buffer, Laemli 2x, Concentrate (#S3401, Sigma Aldrich) and boiled 10 min at 95ºC. An SDS-PAGE electrophoresis was performed following the protocol, and the gel was stained overnight in SimplyBlue™ SafeStain (Thermofisher).
Week 37
Week 38
14/9
Alicia
16/9
Alicia
17/9
Alicia
Week 39
Week 40
Week 41
Week 42