Team:Paris Bettencourt/Notebook/Interlab Study

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July

July 4th

We will participate in the iGEM interlab study

Device 1

Existing device: BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector.

Kit location
• Spring 2014 Plate 4, Well 18A

We use NEB Turbo cells to transform with the Biobrick

Biobrick extraction (iGEM protocol)

To use the DNA in the Distribution Kit you may follow these instructions:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want.
2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.

Then we followed our protocol for Heat Shock transformation of E.coli

July 5th

Bacterias plated yesterday growed and made big and small colonies.I picked one small and one big colony from one plate in order to make liquid culture:

1. 5mL of LB
2. 5um Chlorophenicol
3. Inoculate a single colony

July 6th

I made a glycerol stock from the liquid culture following the standard protocol of the Glycerol stock and labelled it with a number G.22

July 7th

Devices 2 and 3

New device to be built by the iGEM team: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone

Kit locations (Spring 2014)

1. BBa_J23101 (called BBa_K823005 when in pSB1C3): Plate 1, Well 20K
2. BBa_E0240 (in pSB1C3): Plate 2, Well 24B

New device to be built by the iGEM team: BBa_J23115 + BBa_E0240 (B0032-E0040-B0015), must be built in the pSB1C3 backbone

Kit locations (Spring 2014)

1. BBa_J23115 (called BBa_K823012 when in pSB1C3): Plate 1, Well 22I
2. BBa_E0240 (in pSB1C3): Plate 2, Well 24B

I followed iGEM Distribution Kit instructions and Heat Shock transformation of E.coli

July 8th

Successful transformations:
• BBa_E0240
• BBa_K823012

Unsuccessful transformation:
• BBa_K823005

For the successful transformations I followed the Glycerol stock protocol

For the unsuccessful transformation I transformed NEB turbo again with the rest of Biobrick BBa_K823005 following again iGEM Distribution Kit instructions and Heat Shock transformation of E.coli

July 9th

I finished the Glycerol stock protocol and labelled G.23 for BBa_K823012 and G.24 for BBa_E0240

BBa_K823005 successfully transformed! I followed the Glycerol stock protocol

Minipreps
Following the standard protocol for the miniprep
I measured DNA content with the nanodrop.
Digestion analysis: (Clovis's recipe)
- 5 ug plasmid (given 211ng of BBa_E0240 - 15uL // given 387ng of BBa_K823012 - 30uL
- 5 ul FD Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)

Result:DNA wasn't cut. Possible causes:
- wrongly calculated volumes (BBa_K823012 shouldn't be 30uL)
- too short digestion time

July 10th

Finished the Glycerol stock protocol and labelled it G.25

Minipreps Following the standard protocol for the miniprep
I measured DNA content with the nanodrop.
Digestion analysis: (Clovis's recipe)
- 5 ug plasmid
- 5 ul FD Buffer
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)
In the final 30uL of the miniprep of BBa_K823005 I didn't have enough of DNA.

July 11th

Minipreps [yes, again!] Following the standard protocol fot the miniprep
I measured DNA content with the nanodrop.
Digestion analysis: (Clovis's recipe)
- 5 ug plasmid
- 5 ul FD Buff(given 211ng of BBa_E0240 - 21uL // given 144ng and 120ng of BBa_K823012 - 35uL // given 273ng of BBa_K823005 - 20uL
- 2.5 uL SpeI + 2.5 uL PstI (BBa_K823012 and BBa_K823005) / 2.5 uL XbeI + 2.5 uL PstI (BBa_E0240)
- complete with H2O
(Final volume of 50 uL)
Result: The gel showed that I mislabelled the constructs. There were 3 promoters (big and very small fragments)
I will restart the experiment from the Biobricks

Biobricks have been taken from the -20C stock and I followed iGEM Distribution Kit instructions and Heat Shock transformation of E.coli

July 12th

Colonies grew for all 3 Biobricks.
They have been put into a liquid culture in order to put them into the Glycerol stock

July 13th

The Glycerol stock have been finish and it is stocked in my box at -20C

July 14th

Minipreps Following the standard protocol for the miniprep
Result: 2 tubes in the fridge -20C.

July 15th

Goal: Ligate and transform BBa_J23101 + BBa_E0240 and BBa_J23115 + BBa_E0240

For both J23101 and J23115 :
- BBa_E0240(GFP) 5 ul
-BBa_J231011/BBa_J23115 1ul
-Ligase 1ul
-Buffer 4ul
-NF water 9ul
TOTAL 20ul

Incubation: 30min 24°C

The ligation products have been transformed following Heat Shock transformation forE.coli

July 16th

Both transformation showed up to be sucessfull. However, the contruct BBa_J23115 + BBa_E0240 did not seem to be fluorescent (observation through the blue filter)

July 24th

I decided to redo the construct BBa_J23115 + BBa_E0240

I inoculated bacterias from the glycerol stock (BBa_J23115 and BBa_E0240) into the culture 5ml LB overnight in 37C

July 25th

I prepared a minipreps following the standard protocol for the miniprep
Then I digested
-BBa_E0240 with XbeI and PstI to extract the GFP coding part
-BBa_J23115 with SpeI and PstI to extract the promotor with the backbone
I have run the digestion products on the elecrophoresis gel
Result:the bands at around 876 bp for GFP and 2100 bp for the promoter + backbone

I performed gel extraction for the GFP fragment and I used some of the digestion product,which I didn't run on the gel, of promoter+backbone to perform a PCR purification. When the gel was running, I added some AP to that sample to avoid end's sticking.

August

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September

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October

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