Contents 1 Notebook 1.1 Tuesday 18.06.13 1.2 Wednesday 19.06.13 1.3 Thursday 20.06.13 1.4 Friday 21.06.13 1.5 Monday 24.06.13 - Friday 28.06.13 1.6 Monday 01.07.13 1.7 Tuesday 02.07.2013 1.8 Wednesday 03.07.2013 1.9 Thursday 04.07.2013 1.10 Tuesday 09.07.2013 1.11 Wednesday 10.07.2013

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 P_m+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.

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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.

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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 [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].

Sample	Concentration (ng/µl)
mCherry1	5,6
mCherry2	7,3

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Friday 21.06.13

We isolated the DNA from the transformed cells by using the miniprep protocol and measured the concentration by [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf 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

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Monday 24.06.13 - Friday 28.06.13

This week we have established protocols for further work and designed primers for PCR.

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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>).

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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.

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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.

Sample	OD600	Estimated CFU
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 [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].

Sample	Concentration (ng/µl)
RBS	4,91
pTet1	8,99
pTet2	4,39
pBAD1	3,54
pBAD2	9,24

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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.

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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.

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Wednesday 10.07.2013

We ran the PCR again with different parameters. Increased annealing- and elongationtime and a separate program for the backbone. We still only got a band for RFP <partinfo>Bba_E1010</partinfo>. In the prosess of reckecking the primers it was discovered that the primers for RFP are incorrect and that 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.