Notebook
Transformed BioBricks
Tuesday 18.06.13
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.
Wednesday 19.06.13
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.
Thursday 20.06.13
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
|
Friday 21.06.13
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
|
Monday 24.06.13 - Friday 28.06.13
This week we have established protocols for further work and designed primers for PCR.
Monday 01.07.13
We transformed some BioBricks: pBAD promoter (<partinfo>BBa_K206000</partinfo>), RBS (<partinfo>BBa_J61101</partinfo>) and a plasmid backbone (<partinfo>BBa_J01101</partinfo>).
Tuesday 02.07.2013
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, pBAD and the plasmid backbone (pTet) from agar plate to liquid medium and incubated at 37° C.
Wednesday 03.07.2013
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. No dilution of the samples was necessary.
| Sample
| OD600
|
| ER2566
| 0.9805
|
| ara-
| 0.1263
|
| ara+
| 0.0471
|
We isolated the DNA from the transformed cells (with RBS, pBAD and pTet) by using the miniprep protocol and measured the concentration by NanoDrop ND-1000 Spectrophotometer.
| Sample
| Concentration (ng/µl)
|
| RBS
| 4,91
|
| pTet1
| 8,99
|
| pTet2
| 4,39
|
| pBAD1
| 3,54
|
| pBAD2
| 9,24
|
Thursday 04.07.2013
Continuation of the small-scale vesicle preparation according to the vesicle isolation protocol. Step 8-12 was completed with the exception that the pellet was resuspended in 0.5 mL DPBSS insted of 100 µL in step 11 and that the check for sterility in step 12 was not performed. The SDS-PAGE showed no signs of vesicles.
Monday 08.07.10
An overnight culture was started for vesicle isolation as desribed for tuesday 02.07.10.
Tuesday 09.07.2013
Today we ran PCR on 4 FP's <partinfo>Bba_K864100</partinfo>, <partinfo>Bba_K592100</partinfo>, <partinfo>Bba_E0040</partinfo>, <partinfo>Bba_E1010</partinfo> and 1 backbone <partinfo>Bba_J01101</partinfo>. We used the Phusion DNA Polymerase protocol from New England Biolabs [1] for 50 ul sample(without DMSO). All amplicons were run on a GelGreen 0.8% agarose gel. We only got a band on our RFP <partinfo>Bba_E1010</partinfo>, and it was not the expected size. We will adjust the PCR parameters and run them again tomorrow.
Step 2-11 of small scale-vesicle preparation protocol (vesicle isolation) and step 1-4 of the density gradient protocol (vesicle isolation) was performed. Samples from the small-scale vesicle preparation were made ready for SDS-PAGE.
The incubation period was revised from the first attempt to isolate vesicles. The duration and other condition is summerised in the table below.
|
| Sample
| Sample
| Sample
|
| Time (h:min)
| ER2566
| MW27784 ara-
| MW27784 ara+
|
| 0:00
|
| Add 250 μL Amp (100X) and 2,5 mL of overnight culture to 250 mL of LB. Incubate at 37 C
| Add 250 μL Amp (100X) and 2,5 mL of overnight culture to 250 mL of LB. Incubate at 37 C
|
| 1:00
| Add 1 mL of overnight culture to 250 mL of LB. Incubate at 37 C
|
|
|
| 2:00
|
|
| Add 1,25 μL arabinose (1000X)
|
| 4:00
| Measure OD-600
| Measure OD-600
| Measure OD-600
|
Optical denisty (OD) at 600 nm was mesured in step 4 as indicated in the table below. No dilution of the samples was necessary.
| Sample
| OD600
|
| ER2566
| 0.1671
|
| ara-
| 0.6726
|
| ara+
| 0.5968
|
Wednesday 10.07.2013
We ran the PCR again with different parameters. Increased annealing- and elongationtime and a separate program for the backbone due to its larger size. We still only got a band for RFP <partinfo>Bba_E1010</partinfo>. In the prosess of rechecking the primers it was discovered that the primers for RFP are incorrect which explains the discrepancy in productsize. The other primers should be working so tomorrow we will redo the dilutions of the primers, increase the elongation time and lower the annealing temperature a little, add DMSO, add a bit larger templatesample and use 1-step PCR.
SDS-PAGE were run with the samples from the vesicle preparaton from the day before. There were no indication of vesicles in the samples.
Step 5 of the purification of vesicles by density gradient was performed. The fraction were freezed down at -80 oC.
Thursday 11.07.2013
Today we ran PCR again. This time we omitted the RFP, since we discovered that one of the primers was incorrect. We diluted the primers one more time and changed the parameters for the PCR. This time we only did one step, with 30 cycles. We also decreased the annealing temperature and increased the elongationtime. The results were more uplifting this time around after running gel electrophoreses (picture coming soon). We got a clear band on the backbone and faint bands on the GFP and SYFP. Since there was a faint band on the backbone of a larger size than the one we wanted, we added 1 µl of Dpn1 to the amplicon and put it in a 37 °C waterbath for an hour. We used the QIAGEN PCR purification kit to isolate the DNA and measured the cioncentration by NanoDrop ND-1000 Spectrophotometer.
| Sample
| Concentration (ng/µl)
|
| SYFP
| 13,16
|
| GFP
| 55,62
|
| BB
| 55,50
|
Sunday 14.07.2013
5 mL cultures of the E.coli strains ER2566, DH5α and MW27784 were incubated in 5 mL LB at 37 oC for 8 h. 2,5 mL of these cell cultures were added to 250 mL of LB and incubated at 37 oC for 13 h.
Monday 15.07.2013
Step 3-11 of small scale-vesicle preparation protocol (vesicle isolation) and step 1-4 of the density gradient protocol (vesicle isolation) was performed. Samples from the small-scale vesicle preparation were made ready for SDS-PAGE.
Optical denisty (OD) at 600 nm was mesured in step 4 as indicated in the table below. The samples were diluted 1:16 with LB media.
| Sample
| OD600
|
| ER2566
| 0.133
|
| DH5alpha;
| 0.169
|
| MW27784
| 0.198
|
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