Team:UNIPV-Pavia/Calendar/July

19° Bio-Lab - I7-3,4 and 5 and I8-3, 4 and 5 were prepared for 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. Other parts were received from Anderson Lab.

20° Bio-Lab - TECAN test provided encouraging results, showing that I7-3, I7-5, I8-4 and I8-5 produced GFP.
Other culture (I7-1, I7-2, I7-4, I8-1, I8-2, I8-3) did not produce GFP, so they were thrown away. A furhter screening were performed on I7-3, I7-5, I8-4, I8-5 and this time all the clones are OK . Inoculum of <partinfo>BBa_J72007</partinfo> and <partinfo>BBa_J72013</partinfo> from LB agar plates.A glycerol stock was prepared for each culture.
Inoculum of <partinfo>BBa_J72008</partinfo> from LB agar plate grown at 30°C 220 rpm overnight.

21° Bio-Lab - Glycerol stock for <partinfo>BBa_J72008</partinfo> was prepared.

22° Bio-Lab - LB agar plates prepared.

23° Bio-Lab - We decided to perform a TECAN test to evaluate the strength of some promoters belonging to the Anderson Promoters Collection.

24° Bio-Lab -Competent cells preparation for:

• MG1655
• BW53474
• BW25141
• BW25142

PCR was performed to amplify Phasins.
11° Meeting - Updating about Bio-Lab activity.

25° Bio-Lab - Transformation of <partinfo>BBa_J23118</partinfo> in our home-made competent strains

• MG1655
• BW25141
• BW25142
• BW53474
• DH5alpha (as control)

to check transformation efficiency.

26° Bio-Lab - All plates showed red colonies, so we calculated the efficiency of the strains.

27° Bio-Lab - Phasins PhaP1 and PhaP2 were amplified by PCR and the results were sequenced .

28° Bio-Lab - Phasins samples were sent to be sequenced. MiniPrep was performed. Ligations (from I11 till I19 and I74c5, I84c5, I104c5) were all performed

29° Bio-Lab - PBHR68 plate showed colonies!!transformation of ligation of yesterday. Screening of <partinfo>BBa_J72007</partinfo> (CRIM), <partinfo>BBa_J72008</partinfo> (helper), pAH123 (helper) ans pCP20 (helper).

30° Bio-Lab -Agar plates after transformation were checked(2 colonies were picked from every plate). MiniPrep was performed for:

• PBHR68 (BioPlastic operon) -> 272 ng/ul
• BBa_J72007(CRIM plasmid) -> 41 ng/ul (not clean spectrum at 230nm)
• BBa_J72013(CRIM plasmid) -> 16 ng/ul

31° Bio-Lab - MiniPrep was performed on 23 falcon tubes containing I11 -> I19 and I7/8/10-4C5 ligations. We decided to performe a TECAN test on our parts, in order to see if RFP was correctly excided from BBa_J231xx vector, since length of RFP and of our insert (RBS-luxI-tt) were similar.

32° Bio-Lab - TECAN test showed that no RFP was produced from our parts.I14-1, I16-1, I17-1, I18-1, I19-1, I74c5-2, I84c5-2 and I12-2 were sequenced. Trasformation of RING into:

• BW25141 (pir+)
• BW25142 (pir116)
• BW23474 (pir116)
• DH5alpha
• MG1655

33° Bio-Lab - Agar plates after transformation were checked (Single colonies were picked from plates). MiniPrep was performed on I10-1, I12-2, I3-1, I14-1, I17-1 and <partinfo>BBa_J23110</partinfo>. Following ligations were performed: I15_new, I14_4C5, I16_4C5, I17_4C5, I18_4C5 and I19_4C5. We incoulated from glycerol stock (3ul in 2ml LB+Amp): <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23118</partinfo>, <partinfo>BBa_J23116</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23106</partinfo>, <partinfo>BBa_J23105</partinfo>, <partinfo>BBa_J23101</partinfo>, <partinfo>BBa_J23100</partinfo>, <partinfo>BBa_B0033</partinfo> in order to perform a TECAN test tomorrow.

34° Bio-Lab -Tecan Test. This morning all the seven cultures were grown, For those cultures glycerol stocks were prepared and stored at -80°C. (Remaning 5 ml were used to perform MiniPrep). Competent cells for cultures inoculated yesterday were prepared. Trasformations of ligations of yesterday were performed.

35° Bio-Lab - Plates were checked. Following ligations were performed: I17_4C5, I18_4C5, I19_4C5, I10-4C5, I12-4C5. Competent T9002 and MC1061 cells were transformed with a negative control in pSB4C5. We also performed a TECAN test in order to evaluate the ranking of sternght of promoters we inoculaed yesterday.

36° Bio-Lab - All plates incubated yesterday (T9002-4C5, MC1051-4C5 and PBHR68-4C5) showed colonies! Trasformations of ligations of yesterday were performed. We also received sequencing results (all correct).

37° Bio-Lab - Two colonies from each plate incubated yesterday were picked. Colonies were counted to evaluate efficiency of transformation for our home made competent cells.

38° Bio-Lab - Screening digestion was performed for colonies picked yesterday. MiniPrep and gel run was performed.

39° Bio-Lab - Ligation of I15_4C5.
12° Meeting - Updating about Bio-Lab activity and organization of our trip to Boston!

40° Bio-Lab - Sample of I15-1 was prepared for sequencing.I14_4C5, I16_4C5, I17_4C5, I18_4C5 and I19_4C5 were cotrasformed in T9002. MiniPrep was performed on MC1, MC2.

41° Bio-Lab - Today we diluted primers to modify PhaPs and performed a PCR with them in order to mutagenize this BioBrick to get its prefix Standard/Silver compliant and suffix Silver compliant.

42° Bio-Lab - PCR was prepared in order to amplify Phasins Phap (<partinfo>BBa_K208001</partinfo>).After that phasins were gel extracted and digested. A tecan test was performed.

43° Bio-Lab - Agar plates were checked and efficiency was calculated. Miniprep of <partinfo>BBa_J04450</partinfo> was performed. Ligation of I20, I21.

44° Bio-Lab - Transformation of I20 and I21 into E. coli DH5-alpha.