Team:UNIPV-Pavia/Calendar/August/settimana3

From 2010.igem.org


AUGUST: WEEK 3



August, 16th

Resuspension from iGEM 2010 Distribution Kit of BBa_J13002 (Plate 1, Well 13B) and BBa_I13522 (Plate 2, Well 8A).

Transformation (1 ul) of BBa_J13002, BBa_I13522, INTEIN, CREAM (Mr.Gene) into 100 ul of E. coli TOP10 home-made competent cells.

Transformation of 15-4C5 (1 ul) into in re-competentized E. coli TOP10 carrying BBa_T9002.

Transfomants were plated on LB+Amp 100 ug/ml agar plates, the last one on LB+Amp100+Cm12,5 agar plates and were let grow ON at 37°C.

August, 17th

All plates showed colonies.

Pick of BBa_J13002, BBa_I13522, INTEIN, CREAM, co-trasformed BBa_T9002 and inoculum into 1 ml of LB+Amp, LB+Kan, LB+Cm, respectively, to make glycerol stocks. Falcons were than refilled for the minipreps of the following day.

Inoculum of I5, I26, BBa_K105012, BBa_B0034, I20M-1, I20A-1, I21M-3, I21-4, I21A-7, I0 for minipreps of the following day.


Cultures were diluted (5ul in 2ml LB+antibiotic) for Tecan Test:

I7-A I8-5 D I9-1
I10-B I12-2 A2
BBa_B0032 I144C5-T9002 I164C5-T9002
I174C5-T9002 I184C5-T9002 I194C5-T9002
ENTERO4C5-T9002

Glycerol stock was prepared for ENTERO4C5-T9002.


August, 18th

Cultures were diluted (5ul in 2ml LB+antibiotic) for Tecan Test:

I7-A I8-5 D I9-1
I10-B I12-2 A2
BBa_B0032 I144C5-T9002 I154C5-T9002
I164C5-T9002 I174C5-T9002 I184C5-T9002
I194C5-T9002 ENTERO4C5-T9002

Glycerol stock was prepared for I154C5-T9002.


The following cultures were miniprepped and quantified with Nanodrop:

  • I26: 67,5 ng/ul
  • BBa_K105012: 60,9 ng/ul
  • BBa_B0034: 64,5 ng/ul
  • BBa_F2620: 91,8 ng/ul
  • I20M-1: 191,8 ng/ul
  • I20A-1: 176,2 ng/ul
  • I21M-3: 156,2 ng/ul
  • I21-4: 76,3 ng/ul
  • I0: 121,2 ng/ul
  • INTEIN: 143,9 ng/ul
  • BBa_J13002: 31,9 ng/ul

and than digested as follows for four hours:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 (ul) Enzyme 2 (ul) Buffer H (ul)
I26 Insert 25 21,5 0 0,5 EcoRI 0,5 SpeI 2,5
BBa_K105012 Vector 25 16,5 5 0,5 E 0,5 S 2,5
BBa_B0034 Vector 25 15,5 6 0,5 E 0,5 S 2,5
BBa_F2620 Insert 25 16,5 5 0,5 E 0,5 S 2,5
I20M-1 Insert/Screening 25 8 13,5 0,5 E 0,5 S 2,5
I20M-1 Insert/Screening 25 8 13,5 0,5 XbaI 0,5 PstI 2,5
I20A-1 Insert/Screening 25 13 8,5 0,5 E 0,5 S 2,5
I20A-1 Insert/Screening 25 13 8,5 0,5 X 0,5 P 2,5
I21-4 Insert/Screening 25 13 8,5 0,5 E 0,5 S 2,5
I21-4 Vector 25 13,1 8,4 0,5 E 0,5 X 2,5
I0 Vector 25 8,2 13,3 0,5 E 0,5 XbaI 2,5
INTEIN Insert 25 10,5 11 0,5 E 0,5 S 2,5
BBa_J13002 Vector 25 21,5 0 0,5 S 0,5 P 2,5

Two medium gel were prepared and samples were loaded and gel run/cut.

Vectors digested and gel run/cut
Inserts digested and gel run/cut

All samples were correctly digested and run; both I20M-1 and I20A-1 are positive, so we decided to use I20M-1; this new screening for I21-4 showed again that it's correct.

Gel extraction gave these results:

  • I0 (E-X): 20,8 ng/ul
  • BBa_F2620 (E-S): 13,5 ng/ul
  • INTEIN (E-S): 6,5 ng/ul
  • BBa_B0034 (E-X): 23,2 ng/ul
  • I20M-1 (E-S): 7,4 ng/ul
  • BBa_J13002 (S-P): 12,5 ng/ul
  • I21-4 (E-S): 7,2 ng/ul
  • I21-4 (E-X): 24,7 ng/ul
  • BBa_K105012 (E-X): 23,1 ng/ul
  • I26 (E-S): 3,4 ng/ul
  • I20M-1 (X-P): 6,5 ng/ul

They were stored ON at -20°C.


August, 19th

Extra E-X digestion (four hours) of previously miniprepped INTEIN

CultureKindH2ODNAEnzyme1Enzyme2Buffer H
INTEIN Vector 13,5 ul7 ul 1 ul EcoRI1 ul XbaI2,5 ul

Sample was loaded into a small agarose gel, run and cut.

After gel estraction was quantified with Nanodrop: 19,6 ng/ul.

Finally were performed the following ligations:

  • I31: I20M-1 (E-S) + BBa_K105012 (E-X)
  • I32: I20M-1 (E-S) + I21-4 (E-X)
  • I33: I20M-1 (E-S) + I0 (E-X)
  • I34: I21-4 (E-S) + I0 (E-X)
  • I35: I21-4 (E-S) + BBa_K105012 (E-X)
  • I36: I21-4 (E-S) + I21-4 (E-X)
  • I37: INTEIN (E-S) + I0 (E-X)
  • I38: BBa_F2620 + BBa_B0034
  • I39: BBa_J13002 + I20M-1 (X-P)
  • I40: I26 (E-S) + INTEIN (E-X)
  • I41: I26 (E-S) + I0 (E-X)

August, 20th

Ligations of previous day were transformed (1 ul) into 100 ul of home-made competent cells 'E. coli DH5-alpha. They were than plated on LB+Amp agar plates and incubated ON at 37°C.


Colony PCR was performed for MyCRIM (9 colonies), 1 blank, 1 negative control (I5(X-S)), 1 positive control (MrGene CRIM from plate).

Screening PCR: MyCRIM (first 9 samples), ladder, blank, RING, CRIM from MrGene.


Tecan Test

August, 21st

Plates with ligations I31..I41 were stored at +4°C. Colonies would have been screened ASAP.


Glycerol stocks and miniprep for MyCrim 1:9. Quantifications:

CultureQuantification (ng/ul)
MyCRIM 1 59
MyCRIM 2 145
MyCRIM 3 108
MyCRIM 4 125
MyCRIM 5 65
MyCRIM 6 72
MyCRIM 7 97
MyCRIM 8 127
MyCRIM 9 151

Digestions were preformed to screen the direction of the fragment CAT-TT-ORI:

DNA Buffer B H2O EcoRI HindIII
2 ul 2.5 ul 18.5 ul 1 ul 1 ul


Screening digestions: MyCRIM (from 1 to 9).


The fragments length confirm us that all the samples has the CAT-TT-ORI fragment ligated in reversed way.