Team:TUDelft/Notebook/Cry
Notebook
Production and characterization of Cry7Ca1
Week 24
Week 25
Week 26
Week 27
Week 28
Week 29
13/7
Alicia & Javier
Transformation of the Cry7Ca1 toxin plasmid: The commercially cloned plasmid containing Cry7Ca1 toxin (pTWIST___Cry7Ca1) was delivered as dry powder (2000 ng). It was resuspended in 50 µl of Nuclease-Free H2O and incubated 5 min at 42ºC to facilitate DNA resuspension. The concentration of DNA measured using a Nanodrop was 44.3 ng/ul. 2 µl of DNA were transformed into 50 µl of BL21 (DE3) Competent E. coli (NEB) following the protocol for heat shock transformation. The plates were incubated overnight at 37ºC.
14/7
Alicia & Javier
Transformation of the Cry7Ca1 toxin plasmid: Colonies were observed in the transformed plates from 13/07/2020. There was no growth in the plate with the negative control. Colonies were observed in the transformed plates from 13/07/2020. There was no growth in the plate with the negative control.
15/7
Gabriela
The overnight cultures grew successfully, and no growth was observed in the negative controls. 3 mini-preps per each colony (colonies 1,2,3) were done following the protocol.
Week 30
22/7
Alicia
Culture for glycerol stock of E. coli BL21 (DE3) Cry7Ca1: A culture of 5 ml LB + 5µl Ampicillin 1000X + 1 colony from the plate from 13-07-2020 was prepared and incubated overnight at 37ºC and 150 rpm.
23/7
Alicia
Culture for glycerol stock of E. coli BL21 (DE3) Cry7Ca1: A glycerol stock was prepared following the protocol. The 5 ml of culture were centrifuged in 1 ml Eppendorf tubes for 10 min at maximum rpm in a table microcentrifuge instead of steps 2-3 of the protocol.
Week 31
28/7
Alicia
Cry7Ca1 expression and bsdBCD decarboxylase expression (0.1 mM IPTG):
The pre-cultures for E. coli BL21 (DE3) pTWIST_Cry7Ca1 and E. coli BL21 (DE3) pTWIST_bsdBCD were prepared by inoculating 5 ml of LB media with 10 µl of Ampicillin 100 mg/ml (1000X), using 10 µl of the preculture from the previous day respectively. The cultures were incubated overnight at 37ºC and 200 rpm.
29/7
Alicia
Cry7Ca1 expression and bsdBCD decarboxylase expression (0.1 mM IPTG): The OD600 of the pre-cultures was measured after an overnight growth and the values were 0.9 and 1.3 for E. coli BL21 (DE3) pTWIST_Cry7Ca1 and E. coli BL21 (DE3) pTWIST_bsdBCD respectively. For each pre-culture, two cultures were done with the following components: 1) 19 ml LB with 20 µl Ampicillin 100 mg/ml (1000X) inoculated with 1 ml of pre-culture; and 2) 18 ml LB with 20 µl Ampicillin 100 mg/ml (1000X) inoculated with 2 ml of pre-culture. The inoculated cultures were incubated at 37ºC and 200 rpm.
The OD600 of each culture was measured over time until it reached 0.4-0.6. Then, 2 ml were taken and the cultures were induced with IPTG 100 mM for a final concentration of 0.1 mM, and incubated at 37 ºC and 200 rpm. The following samples were taken after induction: E. coli BL21 (DE3) pTWIST_Cry7Ca1 induction after 2h and 4h; and E. coli BL21 (DE3) pTWIST_bsdBCD induction after 1h, 3h and 4h. For each timepoint measured, the OD was measured and samples were taken, corresponding to an OD600 1-1.2 to normalize per number of cells. All the samples taken were spin down 10 min at maximum rpm and stored at -20ºC until further use.
30/7
Alicia
Cry7Ca1 expression and bsdBCD decarboxylase expression (0.1 mM IPTG): After an overnight incubation, the OD was measured and samples were taken, corresponding to an OD600 1-1.2 to normalize per number of cells. All the samples taken were spin down 10 min at maximum rpm and stored at -20ºC until further use.
After an overnight incubation, the OD was measured and samples were taken, corresponding to an OD600 1-1.2 to normalize per number of cells. All the samples taken were spin down 10 min at maximum rpm and stored at -20ºC until further use.
Week 32
3/8
Alicia
Cry7Ca1 expression and bsdBCD decarboxylase expression. Sample preparation:
Frozen pellets from 29-07-2020 and 30-07-2020 were unfrozen and resuspended in 100 µl of Nuclease-Free H2O. Only the first replicate of each E. coli BL21 (DE3) pTWIST_Cry7Ca1 and E. coli BL21 (DE3) pTWIST_bsdBCD was used for further analysis. The following samples were used: E. coli BL21 (DE3) pTWIST_Cry7Ca1 pre-induction (PI), induction after 2h, 4h and overnight; and E. coli BL21 (DE3) pTWIST_bsdBCD pre-induction (PI), induction after 1h, 3h, 4h and overnight. 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. Boiled samples were stored in the freezer at -20ºC.
4/8
Alicia
Cry7Ca1 expression and bsdBCD decarboxylase expression. SDS-PAGE electrophoresis: Samples prepared on 3-08-2020 were unfrozen, and 5 µl of Nuclease-free H2O and 5 µl of Sample Buffer, Laemli 2x, Concentrate (#S3401, Sigma Aldrich) were added to increase the sample volume.
An SDS-PAGE electrophoresis was performed following the protocol, and the gel was stained overnight in SimplyBlue™ SafeStain (Thermofisher).
The second gel was used to perform Western blot following the protocol.
Week 33
12/8
Alicia
Cry7Ca1 expression (0.2 and 0.5 mM IPTG): The pre-cultures for E. coli BL21 (DE3) pTWIST_Cry7Ca1 and E. coli BL21 (DE3) pTWIST_bsdBCD were prepared by inoculating 5 ml of LB media with 10 µl of Ampicillin 100 mg/ml (1000X), using the corresponding glycerol stocks. A pre-culture of E. coli BL21 (DE3) was prepared by inoculating 5 ml of LB media from a glycerol stock was used as a control. The cultures were incubated overnight at 37ºC and 150 rpm.
13/8
Alicia
Alicia: Cry7Ca1 expression (0.2 and 0.5 mM IPTG):
The OD600 of the pre-cultures was measured after an overnight growth and the values were 1.6, 1.7 and 1.7 for E. coli BL21 (DE3) (control), E. coli BL21 (DE3) pTWIST_Cry7Ca1 and E. coli BL21 (DE3) pTWIST_bsdBCD respectively. For each pre-culture, three cultures were done with the following components: 19 ml LB with 20 µl Ampicillin 100 mg/ml (1000X) inoculated with 1 ml of pre-culture (the control did not have Ampicillin). The inoculated cultures were incubated at 37ºC and 200 rpm.
The OD600 of each culture was measured over time. As the E. coli BL21 (DE3) pTWIST_bsdBCD grew slower than the rest of the cultures, these cultures were discarded. When the cultures reached a OD of 0.77-0.79 for the control and 0.58-0.59 for the E. coli BL21 (DE3) pTWIST_Cry7Ca1 cultures, 2 ml of each culture were taken as pre-induction sample. From that sample, 1.3 ml and 1.7 ml respectively were taken to normalize the number of cells for an OD600 of 1, and were centrifuged 10 min at maximum rpm. The supernatant was discarded, and the samples stored at -20ºC until further use.
The cultures were induced with IPTG 100 mM for a final concentration of 0, 0.2 and 0.5 mM IPTG and incubated at 30 ºC and 200 rpm. After 2h and 4h of induction, the OD600 was measured and samples were taken, corresponding to an OD600 1 to normalize per number of cells. All the samples taken were spin down 10 min at maximum rpm and stored at -20ºC until further use.
14/8
Alicia
Cry7Ca1 expression (0.2 and 0.5 mM IPTG) : After an overnight incubation, the OD was measured and samples were taken, corresponding to an OD600 1 to normalize per number of cells. All the samples taken were spin down 10 min at maximum rpm, the supernatant was discarded and the pellets stored at -20ºC until further use.
Week 34
Week 35
Week 36
3/9
Alicia
Cry7Ca1 protein expression: pre-cultures:
Maartje:
Pre-cultures for Cry7Ca1 expression 0.5 and 1 mM IPTG
The pre-cultures for E. coli BL21 (DE3) pTWIST_Cry7Ca1 was prepared by inoculating 15 ml of LB media with 15 µl of Ampicillin 100 mg/ml (1000X), using the corresponding glycerol stock. A pre-culture of E. coli BL21 (DE3) was prepared by inoculating 15 ml of LB media from a glycerol stock was used as a control. The cultures were incubated overnight at 37ºC and 200 rpm.
Week 37
4/9
Alicia
Cry7Ca1 overexpression (0.5 and 1 mM IPTG): The OD600 of the pre-cultures was measured after an overnight growth and the values were 3 and 3.45 for E. coli BL21 (DE3) (control) and E. coli BL21 (DE3) pTWIST_Cry7Ca1 respectively. For each pre-culture, three cultures were done with the following components: 19.7 ml LB with 20 µl Ampicillin 100 mg/ml (1000X) inoculated with 300 µl of pre-culture (the control did not have Ampicillin).
The OD600 of each culture was measured over time. When the cultures reached an OD of ̴ 0.4 -0.6, 2 ml of each culture were taken as pre-induction sample. As the control cultures grew faster, they were kept at 4ºC until the other cultures reached the target OD600. From the pre-induction samples taken, 1.2 ml/sample were taken to normalize the number of cells for an OD600 of 1. These samples were centrifuged 10 min at maximum rpm. The supernatant was discarded, and the samples stored at -20ºC until further use.
The OD600 of each culture was measured over time. When the cultures reached an OD of ̴ 0.4 -0.6, 2 ml of each culture were taken as pre-induction sample. As the control cultures grew faster, they were kept at 4ºC until the other cultures reached the target OD600. From the pre-induction samples taken, 1.2 ml/sample were taken to normalize the number of cells for an OD600 of 1. These samples were centrifuged 10 min at maximum rpm. The supernatant was discarded, and the samples stored at -20ºC until further use.
5/9
Alicia
Cry7Ca1 expression (0.5 and 1 mM IPTG): After an overnight incubation, the OD was measured and samples were taken, corresponding to an OD600 1 to normalize per number of cells. All the samples taken were spin down 10 min at maximum rpm, the supernatant was discarded, and the pellets stored at -80ºC until further use.
Week 38
14/9
Alicia
Cry7Ca1 expression and purification (0.5 mM IPTG):The pre-cultures for E. coli BL21 (DE3) pTWIST_Cry7Ca1 was prepared by inoculating 10 ml of LB media with 10 µl of Ampicillin 100 mg/ml (1000X), using the corresponding glycerol stock. A pre-culture of E. coli BL21 (DE3) was prepared by inoculating 10 ml of LB media from a glycerol stock was used as a control. The cultures were incubated overnight at 37ºC and 200 rpm.
15/9
Alicia
Cry7Ca1 expression and purification (0.5 mM IPTG): The OD600 of the pre-cultures was measured after an overnight growth and the values were 2.5 and 2.48 for E. coli BL21 (DE3) (control) and E. coli BL21 (DE3) pTWIST_Cry7Ca1 respectively. For E. coli BL21 (DE3) (control) pre-culture, two cultures were done with the following components: 19.6 ml LB inoculated with 400 µl of pre-culture. For E. coli BL21 (DE3) pTWIST_Cry7Ca1 pre-culture, two cultures were done with the following components: 19.6 ml LB with 20 µl Ampicillin 100 mg/ml (1000X) inoculated with 350 µl of pre-culture. The inoculated cultures were incubated at 37ºC and 200 rpm.
The OD600 of each culture was measured over time. As the cultures were too grown, the experiment was stopped. These cultures were used to prepare pre-cultures for the following day. The pre-cultures for E. coli BL21 (DE3) pTWIST_Cry7Ca1 was prepared by inoculating 10 ml of LB media with 10 µl of Ampicillin 100 mg/ml (1000X), with 100 µl of the corresponding growing culture. A pre-culture of E. coli BL21 (DE3) was prepared by inoculating 10 ml of LB media with 100 µl of the corresponding growing culture was used as a control. The cultures were incubated overnight at 37ºC and 200 rpm.
16/9
Alicia
Cry7Ca1 expression and purification (0.5 mM IPTG): The OD600 of the pre-cultures was measured after an overnight growth and the values were 3.1 and 4.1 for E. coli BL21 (DE3) (control) and E. coli BL21 (DE3) pTWIST_Cry7Ca1 respectively. For E. coli BL21 (DE3) (control) pre-culture, one culture was done with the following components: 19.6 ml LB inoculated with 320 µl of pre-culture. For E. coli BL21 (DE3) pTWIST_Cry7Ca1 pre-culture, one culture was done with the following components: 19.6 ml LB with 20 µl Ampicillin 100 mg/ml (1000X) inoculated with 240 µl of pre-culture. The inoculated cultures were incubated at 37ºC and 200 rpm.
The OD600 of each culture was measured over time. When the cultures reached an OD of ̴ 0.4 -0.6, 2 ml of each culture were taken as pre-induction sample. As the control cultures grew faster, they were kept at 4ºC until the other cultures reached the target OD600. From the pre-induction samples taken, 1.2 ml/sample were taken to normalize the number of cells for an OD600 of 1. These samples were centrifuged 10 min at maximum rpm. The supernatant was discarded, and the samples stored at -80ºC until further use.
The OD600 of each culture was measured over time. When the cultures reached an OD of ̴ 0.4 -0.6, 2 ml of each culture were taken as pre-induction sample. As the control cultures grew faster, they were kept at 4ºC until the other cultures reached the target OD600. From the pre-induction samples taken, 1.2 ml/sample were taken to normalize the number of cells for an OD600 of 1. These samples were centrifuged 10 min at maximum rpm. The supernatant was discarded, and the samples stored at -80ºC until further use.
17/9
Alicia
Cry7Ca1 expression and purification (0.5 mM IPTG): After an overnight incubation, the OD was measured and samples were taken, corresponding to an OD600 1 to normalize per number of cells. All the samples taken were spin down 10 min at maximum rpm, the supernatant was discarded, and the pellets stored at -80ºC until further use. A sample of 700 µl was also taken for protein purification.
Samples taken for purification were used, and Cry7Ca1 was purified following the protocol.
SDS-PAGE electrophoresis:
Week 39
Week 40
Week 41
Week 42