Team:Goettingen/Notebook/reporterSystems July

Again transformations

02.07.18

  • Transformation of pIGEM2, pIGEM2, pIGEM3, and pIGEM5 into E. coli DH5α to geht backup plasmids.
  • Transformation of SP1 with pIGEM5.

Evaluation of transformations

03.07.18

DH5α transformants were separated. SP1-pIGEM2/3 transformants were also separated. The strain SP1 was transformed with pBQ200 to create an isogenic control. For this transformation, we used 2 µL plasmid. The following table shows the results from the transformations.

Strain Cfu (normal/rest)
DH5α_pIGEM1 137/960
DH5α_pIGEM2 1072/1450
DH5α_pIGEM3 36/204
DH5α_pIGEM5 616/1096
SP1_pIGEM3 43/94
SP1_pIGEM5 –/–

Plasmid preparation and preparation of "modified disc assay"

04.07.18

The plasmid pIGEM5 was isolated from E. coli with the normal protocol. Concentration: 141.4 ng/µL.
A PCR was performed with the primers ML107 and lacZ-rev on the plasmid to check if there is an insert. This PCR was succesful.
The transformations of SP1 with pBQ200 were separated.
CS-Glu agar with and w/o tryptophan were prepared. In the middle of the plate was a circle of the agar extracted with an open falcon tube. The precultures for this experiment (SP1_pIGEM3) were incubated over night in LB medium at 28°C and agitation.

Furthermore was the β-galactosidase assay prepared for the next day with the strains:

Strain Origin Genotype
GP342 168 derivative trpC2 amyE::(PgltA-lacZ aphaA3)
GP650 GP342 derivative trpC2 amyE::(PgltA-lacZ aphaA3) gltC::Tn10 spc
iGEM20 cDNA GP342→SP1 amyE::(PgltA-lacZ aphaA3)
iGEM21 cDNA GP650→SP1 amyE::(PgltA-lacZ aphaA3) gltC::Tn10 spc

05.07.18

Modified disc assay

Cells from the over night culture were propagated on CS-Glu agar containing X-Gal, kanamycin and NO tryptophan. Into the holes in the plates, we filled 400 µL CS-Glu medium and 400 µL CS-Glu medium with 50 mM glyphosate. The plates were incubated over night at 37°C.

The transformation of SP1 with pIGEM5 was repeated.

Preparation of β-galactosidase assay

The strains iGEM20 and SP1_pIGEM3 were used. Some colonies were picked from the plates and resuspended in 50 µL LB medium. 5 µL were transferred into 4 mL LB medium and the remaining 45 µL were also transferred into 4 mL LB medium. The cells were incubated over daytime and then was CS-Glu medium (5 mL) inoculated with the following pattern.

A B C D
OD600 0.05 0.05 1/10 cell amount of A/B 0.1
Temperature 37°C 28°C 37°C 37°C

β-galactosidase assay

06.07.18

With the overnight cultures were 10 mL CS-Glu medium inoculated to OD600=0.1, one time with 0.75 mM glyphosate and ones without.

Strain OD600 Time
SP1_pIGEM3 –GS 0.51 6 h
SP1_pIGEM3 +GS 0.496 6 h
iGEM20 +GS 0.235 6 h

The cells were harvested with the following protocol:

  • 1.5 mL SP1_pIGEM3 with and w/o glyphosate were centrifuged for 10 min at 5,000 rpm and 4°C.
  • 4.5 mL iGEM20 with glyphosate was centrifuged for 10 min at 5,000 rpm and 4°C.

09.07.18

Transformation of SP1 with pIGEM5 was performed like in protocol described.

Precultures of strain iGEM21 in 5 mL CS-Glu w/o tryptophan and strain iGEM3 in 5 mL CS-Glu w/o tryptophan were prepared and incubated over night at 37°C with agitation.

β–galactosidase assay

10.07.18

For the β-galactosidase assay, SP1_pIGEM3 and GP342 were transferred into 4 mL LB medium and incubated over night.
For the modified disc assay, 4 mL LB medium were inoculated with SP1_pIGEM3.
Primers for all biobricks were designed.

β–galactosidase assay
Strain iGEM3
GS concentration [mM] OD600 after 5.5 h
0 0.592
0.5 0.592
1 0.512
1.5 0.366
2 0.286
Strain GP342
GS concentration [mM] OD600 after 5.5 h
0 0.64
0.5 0.696
1 0.632
1.5 0.392
2 0.346

Plasmid preparation of pBQ200 and pAC7. Furthermore, a chromosomal DNA isolation was performed for Bacillus strains 168 and SP1.

Again β–galactosidase assay

11.07.18

Over night cultures were transferred into CS-Glu medium to an OD600=0.1. The medium contained 0, 0.5, 1, 1.5, 2, mM glyphosate. After 8 h incubation, the following OD600 values were measured.

OD600 of strain 0 mM GS 0.5 mM GS 1 mM GS 1.5 mM GS 2 mM GS
SP1_pIGEM3 0.584 0.584 0.512 0.366 0.286
GP342 0.64 0.696 0.632 0.392 0.346

The modified disc assay was repeated.

β–galactosidase assay

12.07.18

The β–galactosidase assay was performed as described previously. The results are shown in the table below.

Sample Δt A420 A595 MU/mg protein
pIGEM3 0 mM 31:30 min 0.075 0.132 36.07
pIGEM3 0.5 mM 32 min 0.071 0.153 29.46
pIGEM3 1 mM 31:30 min 0.047 0.094 31.74
pIGEM3 1.5 mM 31:30 min 0.025 0.049 32.39
pIGEM3 2 mM 31:30 min 0.014 0.027 32.92
GP342 0 mM 7:30 min 0.392 0.134 780
GP342 0.5 mM 7 min 0.316 0.172 524.9
GP342 1 mM 7 min 0.316 0.118 765.13
GP342 1.5 mM 16 min 0.198 0.066 375
GP342 2 mM 26:30 min 0.115 0.03 289.3
pIGEM3 0 mM 29 min 0.043 0.069 42,9
pIGEM3 0.75 mM 29 min 0.04 0.051 54
GP 342 0 mM 29 min 0.045 0.002 1551
Control 29 min – – –

New β–galactosidase assay

31.07.18

The following table shows the samples for the β–galactosidase assay.

Sample Tryptophane Glyphosate
1 100 µL –
2 100 µL 1 mM
3 50 µL –
4 50 µL 1 mM
5 25 µL –
6 25 µL 1 mM
7 – –
8 – 1 mM

After 5 h, the cells with the following OD600 were harvested (1.5 mL) with 13,000 rpm for 10 min at 4°C.

Sample OD600 after 5 h
1 0.57
2 0.5
3 0.59
4 0.5
5 0.59
6 0.48
7 0.61
8 0.51