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JULY: WEEK 1
June, 28th
Inoculum from glycerol stock for I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 in 5ml LB+Amp. Cultures were grown ON at 37°C 220rpm.
Inoculum of PhaP-1 and PhaP-2 in 5ml LB+Kan and ON growth at 37°C 220rpm.
We received strains and plasmid from CGSC (Yale University)
| Culture name | description | growth conditions that will be used
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| BT340 | pCP20 plasmid | LB+Amp LC 30°C,LB+Amp HC 30°C
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| BW23473 | E. coli pir+ strain | LB, 37°C
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| BW23474 | E. coli pir116 strain | LB, 37°C
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| BW25141 | E. coli pir+ strain | LB, 37°C
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| BW25142 | E. coli pir116 strain | LB, 37°C
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| BW5328 /pAH123 | helper plasmid | LB+Amp LC 30°C,LB+Amp HC 30°C
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| MC1061 | E. coli strain for integration | LB, 37°C
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| MG1655 | E. coli wild type strain | LB, 37°C
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June, 29th
All cultures were grown. From I7-1, I7-2, I8-1, I8-2, I9-1, I9-2, I10-1 and I10-2 5ul were aliquoted and diluted in 500ul LB+Amp for a preliminary TECAN test.
Cultures were MiniPrepped with the following quantifications (NanoDrop):
| Culture name | quantification
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| I7-1 | 83,3 ng/ul
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| I7-2 | 104 ng/ul
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| I8-1 | 123,8 ng/ul
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| I8-2 | 103,7 ng/ul
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| I9-1 | 117,8 ng/ul
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| I9-2 | 110,4 ng/ul
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| I10-1 | 98,8 ng/ul
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| I10-2 | 93,6 ng/ul
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| PhaP-1 | 29,1 ng/ul
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| PhaP-2 | 13,6 ng/ul
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All cultures, except PhaP-1 and PhaP-2 were digested. Digestion of:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 | Enzyme 2 | Buffer H
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| I7-1 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I7-2 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I8-1 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I8-2 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I9-1 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I9-2 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I10-1 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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| I10-2 | Screening | 25 | 2 | 19,5 | 0,5 EcoRI | 0,5 PstI | 2,5
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Digestion was performed at 37°C for 3 hours. Digestions were gel run. For I9 and I10 clones all colonie were correct, while for I7 and I8 extra bands could be observed. Since preliminar results of TECAN test didn't provide encouraging results, we decided to select other 3 colonies both for I7 and I8 and to repeat the screening, while I9 and I10 were correct at this screening, so we chose I9-1 and I10-1 as definitive I9 and I10 parts. I9 and I10 will be further screened next week.
TECAN TEST
 Screening of I7, I8, I9, I10
PhaP-1 and PhaP-2 were prepared or sequencing: 13,8 ul (2x quantity) of PhaP-1 and 14,7ul (1x quantity) oh PhaP-2 were dryed at 65°C and setn to BMR genomics for sequencing service.
All strains received from Yale Univesity were on blotting paper disks, that were placed on an LB agar plates (with/without antiobiotic, see growth conditions), resuspended wih 80ul LB and then streaked. After plate streaking, disks were transferred in falcon tubes containing liquid LB.
- BT340 was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml).
- BW5328 was streaked on LB+Amp (50ng/ml) agar plates and from here on LB+Amp (100ng/ml) agar plates. The paper disk was then transferred in liquid LB+Amp (100ng/ml).
- MG1655 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
- MC1061 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
- BW25142 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
- BW25141 was streaked on LB agar plates. The paper disk was then transferred in liquid LB.
June, 30th
Inoculum of cultures for a TECAN test. From glycerol stocks: I7-1, I7-2, I8-1, I8-2 (all tese 4 colonies were not correct! we want to see if any fluorescence is observed in order to understamd if there is any unexpected GFP production), I9-1, I9-2, I10-1, I10-2. For these cultures 8ul of glycerol stock were inoculated in 2 ml Lb+Amp.
Other three colonies were peaked from I7 and I8 plates.
| I7-3 | 5 ml LB+ Amp, grown ON 37°C 220 rpm
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| I7-4 | 5 ml LB+ Amp, grown ON 37°C 220 rpm
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| I7-5 | 5 ml LB+ Amp, grown ON 37°C 220 rpm
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| I8-3 | 5 ml LB+ Amp, grown ON 37°C 220 rpm
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| I8-4 | 5 ml LB+ Amp, grown ON 37°C 220 rpm
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| I8-5 | 5 ml LB+ Amp, grown ON 37°C 220 rpm
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All cultures were incubated ON, 37°C 220 rpm.
July, 1st
First TECAN test
All cultures incubated yesterday night were diluted 1:100 (10ul in 1ml fresh LB+Amp) and let grown for further 3 hours at 37°C 220 rpm. Also I7-3,4 and 5 and I8-3, 4 and 5 were prepared for TECAN test (10ul were diluted in 1ml LB+Amp). After dilution, static OD was measured for these cultures and a proper dilution was perfeormed, in order to start from the desired OD of 0.02, according to the formula:
After dilution, 3 aliquotes each of 200ul were transferred in a 96-wells plate and incubated in TECAN multi-well plate reader. Cultures were incubated at 37°C and a kinetic cycle was performed:
- Duration: 20h
- Kinetic cycle: 5 minutes
- Every 5 minutes:
- 15 s shaking (3mm, linear)
- 10 s wait
- OD600 measurement
- GFP measurement
I7-3, I7-4, I7-5, I8-3, I8-4 and I8-5 cultures were prepared in 5ml LB+Amp, so the remaining cultures not used for TECAN test were pelletted at 4000 rpm for 10 minutes, supernatant was discarded and pelletes were stored at 4°C for tomorrow MiniPrep of cultures that provided promising results at TECAN test.
Of the plates incubated yesterday, a colony was picked and grown in 1ml LB (+ antibiotic) for 6 hours at 37°C 220 rpm:
| Culture name | Growth condition
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| MG1655 | 1ml LB
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| MG1655 BUP (backup) | 1ml LB
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| MC1061 | 1ml LB
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| MC1061 BUP | 1ml LB
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| BW23142 | 1ml LB
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| BW23142 BUP | 1ml LB
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| BW23141 | 1ml LB
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| BW23141 BUP | 1ml LB
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Other parts were received from Anderson Lab. in stab form.
| Part | Description | Growt condition
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| <partinfo>BBa_J72007</partinfo> | BamHI methyltransferase encoding CRIM plasmid | Streaked on LB+Amp (50ng/ml) agar plate
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| <partinfo>BBa_J72008</partinfo> | phi80 integration helper plasmid pInt80-649 | Streaked on LB+Amp(100ng/ml)+Cm(12,5 ng/ml) agar plate and on LB+Cm (34 ng/ml) agar plate
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| <partinfo>BBa_J72013</partinfo> | BglII methyltransferase encoding CRIM plasmid | Streaked on LB+Cm (34ng/ml) agar plate
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Plates were incubated at 37°C ON.
July, 2nd
TECAN test provided encouraging results, showing that I7-3, I7-5, I8-4 and I8-5 produced GFP. Also I9-1, I9-2, I10-1 and I10-2 confirmed the preliminar results of the precious test, in fact GFP production was observe. At a preliminary analysis, it seems that GFP production is correlated to the srength of the promoter regulating luxI production!! :)
Other culture (I7-1, I7-2, I7-4, I8-1, I8-2, I8-3) did not produce GFP, se they were thrown away.
A furhter screening were performed on I7-3, I7-5, I8-4, I8-5, I9-1 and I10-1.
For this reason, MiniPro was performed for the following colonies, with the following quantiications:
| I7-3 | 107,7 ng/ul
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| I7-5 | 55,4 ng/ul
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| I8-4 | 94,5 ng/ul
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| I8-5 | 118,3 ng/ul
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I7-3, I7-5, I8-4 and I8-5 were digested with EcorI and PstI. All colonies were digested with NheI-PstI (2 NheI sites are present in the BBa_J23xx promoter, so this digestion shows if the promoter has been ligated.)
Digestion of:
| Culture | Kind | Final reaction volume (ul) | DNA (ul) | H20 (ul) | Enzyme 1 | Enzyme 2 | Buffer H
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| I7-3 | Screening | 25 | 2 | 18,5 | 1 EcoRI | 1 PstI | 2,5
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| I7-5 | Screening | 25 | 2 | 18,5 | 1 EcoRI | 1 PstI | 2,5
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| I8-4 | Screening | 25 | 2 | 18,5 | 1 EcoRI | 1 PstI | 2,5
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| I8-5 | Screening | 25 | 2 | 18,5 | 1 EcoRI | 1 PstI | 2,5
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| I7-3 | Screening | 25 | 2 | 18,5 | 1 NheI | 1 PstI | 2,5
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| I7-5 | Screening | 25 | 2 | 18,5 | 1 NheI | 1 PstI | 2,5
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| I8-4 | Screening | 25 | 2 | 18,5 | 1 NheI | 1 PstI | 2,5
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| I8-5 | Screening | 25 | 2 | 18,5 | 1 NheI | 1 PstI | 2,5
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| I9-1 | Screening | 25 | 2 | 18,5 | 1 NheI | 1 PstI | 2,5
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| I10-1 | Screening | 25 | 2 | 18,5 | 1 NheI | 1 PstI | 2,5
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Digestions were incubated at 37°C for 3 hours and gel run.
 Screening of new colonies of I7 and I8. New screening for I9, I10 This time all the clones are OK :)
We choose the following colonies:
| Colony chosen | ligation name
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| I7-3 | I7
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| I8-5 | I8
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| I9-1 | I9
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| I10-1 | I10
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Inoculum of <partinfo>BBa_J72007</partinfo> and <partinfo>BBa_J72013</partinfo> from LB agar plates: one colony from each plte in 1ml LB+Cm (34) at 37°C 220 rpm for 7 hours. Glycero, stock was prepared for each culture.
Inoculum of <partinfo>BBa_J72008</partinfo> from LB agar plate (one colony in 3ml LB+Amp 50), grown at 30°C 220 rpm overnight.
July, 3rd
Glycerol stock for <partinfo>BBa_J72008</partinfo> was prepared.
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