Team:Aalto-Helsinki/InterLab
Introduction
Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values.
Results
Competency
Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 108 to 6 ⋅ 108 cfu/µg. The transformation efficiency was 6.6 ⋅ 105 cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.
Parts
The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter.
Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values.
Results
Competency
Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 108 to 6 ⋅ 108 cfu/µg. The transformation efficiency was 6.6 ⋅ 105 cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.
| DNA concentration [pg/µl] | 10 | 100 |
|---|---|---|
| Plate 1 | 10 | 51 |
| Plate 2 | 5 | 89 |
| Plate 3 | 3 | 76 |
| Average | 6 | 72 |
| DNA concentration [pg/µl] | 10 | 100 |
|---|---|---|
| Plate 1 | 1 ⋅ 106 cfu/µg | 5.1 ⋅ 105 cfu/µg |
| Plate 2 | 5 ⋅ 105 cfu/µg | 8.9 ⋅ 105 cfu/µg |
| Plate 3 | 3 ⋅ 105 cfu/µg | 7.6 ⋅ 105 cfu/µg |
| Average | 6 ⋅ 105 cfu/µg | 7.2 ⋅ 105 cfu/µg |
Parts
The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter.
| Device | Part Number | |
|---|---|---|
| Negative control | BBa_R0040 | |
| Positive control | BBa_I20270 | |
| Test Device 1 | BBa_J364000 | |
| Test Device 2 | BBa_J364001 | |
| Test Device 3 | BBa_J364002 | |
| Test Device 4 | BBa_J364007 | |
| Test Device 5 | BBa_J364008 | |
| Test Device 6 | BBa_J364009 |
| LUDOX CL-X | ddH2O | |
|---|---|---|
| Replicate 1 | 0.051 | 0.041 |
| Replicate 2 | 0.051 | 0.036 |
| Replicate 3 | 0.048 | 0.036 |
| Replicate 4 | 0.060 | 0.038 |
| Arith. Mean | 0.053 | 0.038 |
| Corrected Abs600 | 0.015 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 4.271 |
