Contents |
Started transforming BioBricks. Transformed some composite BioBricks consisting of constitutive promotor, RBS, a fluorescent protein and a terminator. Transformed the parts Promoter+RBS+RFP+term (<partinfo>BBa_I13521</partinfo>), Promoter+RBS+GFP+term (<partinfo>BBa_I13522</partinfo>), Promoter+RBS+CFP+term (<partinfo>BBa_I13600</partinfo>) and Pm+RBS+mCherry+term to competent E.coli DH5α cells, to have some colorful cells to show the camera crew from NRK (Norwegian broadcasting), who will visit our lab tomorrow :-)
For our transformation protocol, see the protocol page.
We transformed BioBricks consisting of fluorescent proteins. Transformed the parts YFP (<partinfo>BBa_E0030</partinfo>), CFP (<partinfo>BBa_E0020</partinfo>), RFP (<partinfo>BBa_E1010</partinfo>), GFP (<partinfo>BBa_E0040</partinfo>), BFP (<partinfo>BBa_K592100</partinfo>) and SYFP (<partinfo>BBa_K864100</partinfo>).
We transferred mCherry from agar plate to liquid medium and incubated at 37° C.
We transferred the transformed cells from yesterday to liquid medium and incubated at 37°C protocol.
We isolated the DNA from the transformed mCherry cells by using the miniprep protocol and measured the concentration by NanoDrop ND-1000 Spectrophotometer.
| Sample | Concentration (ng/µl) |
| mCherry1 | 5,6 |
| mCherry2 | 7,3 |
We isolated the DNA from the transformed cells by using the miniprep protocol and measured the concentration by NanoDrop ND-1000 Spectrophotometer.
| Sample | Concentration (ng/µl) |
| CFP1 | 8,59 |
| CFP2 | 7,04 |
| BFP | 7,06 |
| SYFP | 5,42 |
| RFP1 | 5,04 |
| RFP2 | 7,52 |
| YFP | 6,79 |
| GFP | 5,27 |
This week we have established protocols for further work and designed primers for PCR.
We transformed some BioBricks: pBAD promoter (<partinfo>BBa_K206000</partinfo>), RBS (<partinfo>BBa_J61101</partinfo>) and a plasmid backbone (<partinfo>BBa_J01101</partinfo>).
This day we started making the small scale vesicle preparation according to our vesicle isolation protocol.
Overnight cell cultures of E.coli ER2566 and E.coli BW27784 transformed with the pUM9 plasmid were preprared. The pUM9 plasmid has amp^R induces a stress respons in E.coli when arabinose is present. According to the litterature (link?) stress in E.coli increases vesicle production.
The ER2566 cell were cultured in plain LB, while the BW27784 cells were cultured in LB with ampenicillin (100 µg/mL).
We transferred the samples of RBS, pTet and pBAD from agar plate to liquid medium and incubated at 37° C.
The small-scale vesicle preparation continued with completion of step 2-7 in the vesicle isolation protocol. There where made three samples in step 2; E.coli ER2566 in LB, E.coli BW27784 in LB with ampenicillin (100 µg/mL) (ara-) and E.coli BW27784 in LB with ampenicillin (100 µg/mL) with the addition of arabinose (0.5 %) after 1 hour of incubtion (ara+).
Optical denisty (OD) at 600 nm was mesured as indicated in the table below.
| Sample | OD600 | Estimated CFU |
| ER2566 | 0.9805 | |
| ara- | 0.1263 | |
| ara+ | 0.0471 |
"
