Team:UNIPV-Pavia/Calendar/September/settimana2

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SEPTEMBER: WEEK 2

September, 6th

Inoculum of I47, I48, I49 into 5 ml LB+Amp for TECAN experiment of the following day.

Inoculum of I44-2 and I51-3 into 5 ml LB+Cm34 to evaluate MiniPrep yields.
Inoculum of I44-2 and I51-3 into 5 ml LB+Cm12,5 to evaluate MiniPrep yields and for sequencing.
Inoculum for MiniPrep and sequencing of:
- <partinfo>BBa_J23100</partinfo>_4C5
- <partinfo>BBa_J23101</partinfo>_4C5
- <partinfo>BBa_J23105</partinfo>_4C5
- <partinfo>BBa_J23106</partinfo>_4C5
- <partinfo>BBa_J23110</partinfo>_4C5
- <partinfo>BBa_J23114</partinfo>_4C5
- <partinfo>BBa_J23116</partinfo>_4C5
- <partinfo>BBa_J23118</partinfo>_4C5
- I7-A
- I8-5-D

Inoculum of cultures for TECAN test on self-inducible promoters:

I14-1
I15-1
I16-1
I17-1
I18-1
I19-1
I14_4C5-2
I15_4C5-2
I16_4C5-1
I17_4C5-1
I18_4C5-1
I19_4C5-1
I6-3
I3-1
<partinfo>BBa_B0031</partinfo>

Glycerol stocks of MG43, MC42, MC43. Static TECAN test (absorbance, green fluorescence and red fluorescence) on 200 ul of these cultures.

Cultures were diluted in 4 ml final of LB in order to obtain the same lowest OD with the following formula: culture to remove and substitute with LB=FinalVolume-(lowestOD/measuredOD)*FinalVolume. Centrifuge at 4000 rpm, 25°C for 5 min to pellet the culture, discard the surnatant and resuspend the pellet with 1 mL of M9. Static TECAN test (absorbance, green fluorescence and red fluorescence) on 200 ul of these cultures.

Liquid PCR was performed on MG43 (3 colonies), MC42 (3 colonies), MC43 (3 colonies), MC1061 (negative control) with the following parameters: T_annealing=52°C, cycles composed by 10 min at 94°C, 30 sec at 94°C, 30 sec at 52°C and 4 min at 72°C . This is the result:

PCR results for screening.

MiniPreps of I44-1:5 and I51-1:5 were performed with these results:

Culture	Quantifications (ng/ul)
I44-1	11,1
I44-2	9,9
I44-3	12,3
I44-4	11,4
I44-5	11,4
I51-1	13,6
I51-2	19,4
I51-3	16,5
I51-4	13,3
I51-5	21,8

Probably was used the ethanol buffer without adding before the ethanol, during the wash step and so the quantifications were so low.

Digestion of I44_1:5 and I51_1:5 performed as follow:

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1 (ul)	Enzyme 2 (ul)	Buffer H (ul)
I44-1	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I44-2	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I44-3	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I44-4	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I44-5	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I51-1	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I51-2	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I51-3	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I51-4	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5
I51-5	Screening	25	21,5	0	0,5 EcoRI	0,5 HindIII	2,5

Gel screening shows that all the constructs were ok, so we decided to choose I44-2 and I51-3.

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September, 7th

MiniPrep for:

Culture	Quantifications (ng/ul)
<partinfo>BBa_J23100</partinfo>_4C5	11,8
<partinfo>BBa_J23101</partinfo>_4C5	39,7
<partinfo>BBa_J23105</partinfo>_4C5	25,4
<partinfo>BBa_J23106</partinfo>_4C5	29,7
<partinfo>BBa_J23110</partinfo>_4C5	24,7
<partinfo>BBa_J23114</partinfo>_4C5	15,4
<partinfo>BBa_J23116</partinfo>_4C5	20,7
<partinfo>BBa_J23118</partinfo>_4C5	20,4
I7-A	113,6
I8-5-D	127,6
I44-2 (Cm 34)	63,9
I51-3 (Cm 34)	100,4
I44-2 (Cm 12,5)	68,1
I51-3 (Cm 12,5)	135,4
I47	74,8

Cultures for TECAN test were diluted in the morning (h10:20) 1:100 (40ul in 4ml) and let grown at 37°C 220 rpm till they reach the desired OD.

Trasformation of I42 and I43 in MC008 and MG008 strains, strain DB3.1 without DNA and pSB1C3 RFP in DB3.1 strain. MG42, MC43, MC42, MC43 were plated on LB+Cm34 plates instead DB3.1 on LB+Cm12.5 plates. Integration protocol first day were performed.

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September, 8th

Tecan Test

Inoculum of:

I31
I32
I35
I36
I37
I38
I40
I41
I20

into 5 ml LB + Amp; already available:

I21(E-S)
I26(E-S)
I0(E-X)

for second step of ligation of phasins/intein

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September, 9th

Minipreps were quantified as follows:

I20: 113.5 ng/ul
I31: 76.4 ng/ul
I32: 85.4 ng/ul
I35: 77.2 ng/ul
I36: 83.8 ng/ul
I37: 156.5 ng/ul
I38: 94.4 ng/ul
I40: 556 ng/ul
I41: 148.2 ng/ul

They were digested:

I20: SpeI-PstI
I31: EcoRI-XbaI
I32: E-S
I35: S-P
I36: E-S
I37: E-X; X-P
I38: E-S
I40: E-S
I41: E-X

MG42 and MG43 showed no colonies: probably the reason was a mistake on the choice of the MG008 strain tubes during the trasformations.

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September, 10th

Tecan Test

Screening digestion of I44-2 and I51-3.

Culture	Kind	Final reaction volume (ul)	DNA (ul)	H20 (ul)	Enzyme 1 (ul)	Enzyme 2 (ul)	Buffer (ul)
I44-2	Vector/Screening	25	1,5	19	1 EcoRI	1 PstII	2,5 H
I44-2	Vector/Screening	25	1,5	19	1 EcoRI	1 HindIII	2,5 B
I44-2	Vector/Screening	25	1,5	19	1 NheI	1 NheI	2,5 T
I51-3	Vector/Screening	25	1	19,5	1 EcoRI	1 PstII	2,5 H
I51-3	Vector/Screening	25	1	19,5	1 EcoRI	1 Hind	2,5 B
I51-3	Vector/Screening	25	1	19,5	1 NheI	1 NheI	2,5 T

Check for I44 and I51.

Pipetted 5ul of the liquid cultures MC42, MC43 on LB+Cm12,5 and the culture spots were streaked on the plates. Plates were incubated ON at 43°C.

Gel run/cut for samples of previous day. Gel extraction showed the following quantifications:

I31 (E-X): 21.2 ng/ul
I32 (E-S): 11.3 ng/ul
I35 (S-P): 24.6 ng/ul
I36 (E-S): 12.1 ng/ul
I37 (E-X): 26 ng/ul
I37 (X-P): 12.2 ng/ul
I40 (E-S): 16.3 ng/ul
I41 (E-X): 24.4 ng/ul
I20 (S-P): 30.3 ng/ul

Digestions were ligated in order to create:

I52: I35(S-P)+I37(X-P)
I53: I32(E-S)+I37(E-X)
I54: I36(E-S)+I37(E-X)
I55: I38(E-S)+I20(E-X)
I56: I26(E-S)+I41(E-X)
I57: I40(E-S)+I0(E-X)
I58: I38(E-S)+I31(E-X)

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September, 11th

MC42 and MC43 plates were transfered at -4°C.

I42 and I43 have been transformed in MG008 strain, plated on LB+Cm34 plates and incubated at 30°C for 48 hours.

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