Team:UNICAMP-EMSE Brazil/Notebook/17 September 2011

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May
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2 3 4 5 6 7 8
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16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31
June
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6 7 8 9 10 11 12
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27 28 29 30
July
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25 26 27 28 29 30 31
August
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29 30 31
September
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5 6 7 8 9 10 11
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October
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24 25 26 27 28 29 30
31


Electrophoresis Gel 1, 17/09/2011.
Gel 1: pSB1/ pSB 2/ pSB 3/ pSB 4/ pSB 5/ RBS+IL-12 C4/ RBS+IL-12 C5/ RBS+HlyD C4/ RBS+HlyD C5/ RBS+Tol C C10/ RBS+Tol C C11/ RBS+QseB+RBS+QseC C10/ RBS+QseB+RBS+QseC C11/ RBS+QseB+RBS+QseC C12/
Electrophoresis Gel 2, 17/09/2011.
RBS+SoxS C10/ RBS+SoxS C11/ RBS+SoxS C12/ RBS+SoxS C13/ RBS+HlyB C10/ RBS+HlyB C11/ RBS+HlyB C12/ SoxS+ RBS+HlyB +RBS+HlyD+ RBS+TolC+T C1 1/ SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C1 2/ SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C3/

17 September 2011

Sequences to ship

Quantification and checking digestion of sequences to ship

Objectives:

  1. Quantification of linearized pSB1C3 vector purified from the gel (volume of ladder applied 6 uL; sample: 5 uL DNA +1 uL buffer),
  2. Checking digestion ( EcoRI and PstI) of samples mini-preped 09/15 and 09/16.
  3. Checking digestion of SoxS+HlyB+HlyD +TolC+T C1 1/ SoxS+HlyB+HlyD +TolC+T C1 2/ SoxS+HlyB+HlyD +TolC+T C1 3 (EcoRI and PstI) of samples mini-preped 09/08.

Samples:

  • Gel 1:
  • pSB1/ pSB 2/ pSB 3/ pSB 4/ pSB 5/ RBS+IL-12 C4/ RBS+IL-12 C5/ RBS+HlyD C4/ RBS+HlyD C5/ RBS+Tol C C10/ RBS+Tol C C11/ RBS+QseB+RBS+QseC C10/ RBS+QseB+RBS+QseC C11/ RBS+QseB+RBS+QseC C12/
  • Gel 2:
  • RBS+SoxS C10/ RBS+SoxS C11/ RBS+SoxS C12/ RBS+SoxS C13/ RBS+HlyB C10/ RBS+HlyB C11/ RBS+HlyB C12/ SoxS+ RBS+HlyB +RBS+HlyD+ RBS+TolC+T C1 1/ SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C1 2/ SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C3/
  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%


  • Results:

Construction Total size (gene+vector) Linear vector size Gene size Result
pSB 1 2070 2070 ---- Small concentration, ~ 3 ng/uL
pSB 2 2070 2070 ---- Small concentration ~ 3 ng/uL
pSB 3 2070 2070 ---- Small concentration ~ 3 ng/uL
pSB 4 2070 2070 ---- Small concentration ~ 3 ng/uL
pSB 5 2070 2070 ---- Small concentration ~ 3 ng/uL
RBS+IL-12 C4 3728 2070 1658 Linearized vector OK. No fragment band.
RBS+IL-12 C5 3728 2070 1658 Linearized vector OK. No fragment band.
RBS+HlyD C4 3566 2070 1496 Linearized vector OK. No fragment band.
RBS+HlyD C5 3566 2070 1496 Linearized vector OK. No fragment band.
RBS+Tol C C10 3650 2070 1580 Linearized vector OK. No fragment band.
RBS+Tol C C11 3650 2070 1580 Linearized vector OK. No fragment band.
RBS+QseB+RBS+QseC C10 4156 2070 2086 Linearized vector OK. No fragment band.
RBS+QseB+RBS+QseC C11 4156 2070 2086 Digestion OK. Strange size for fragment band (maybe it is the closed vector band).
RBS+QseB+RBS+QseC C12 4156 2070 2086 Linearized vector OK. No fragment band.
RBS+SoxS C10 2173 2070 103 Linearized vector OK. No fragment band.
RBS+SoxS C11 2173 2070 103 Linearized vector OK. No fragment band.
RBS+SoxS C12 2173 2070 103 Linearized vector OK. No fragment band.
RBS+SoxS C13 2173 2070 103 Linearized vector OK. No fragment band.
RBS+HlyB C10 4253 2070 2183 Linearized vector OK. Closed vector band.
RBS+HlyB C11 4253 2070 2183 Linearized vector OK. No fragment band.
RBS+HlyB C12 4253 2070 2183 Linearized vector OK. Closed vector band.
SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C1 1 8662 3205 5457 Digestion did not work
SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C1 2 8662 3205 5457 Digestion did not work
SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C3 8662 3205 5457 Digestion OK. Fragment band visible. Closed vector band.



Electrophoresis Gel 3, 17/09/2011.
Gel 3: (d)RBS+HlyD C4/ (nd)RBS+HlyD C4/ (d)RBS+Tol C C11/ (nd)RBS+Tol C C11/ (d)RBS+QseB+RBS+QseC C10/ (nd)RBS+QseB+RBS+QseC C10/(d)RBS+SoxS C13/ (nd)RBS+SoxS C13/ (d)RBS+HlyB C10/ (nd)RBS+HlyB C10/ (d)RBS+HlyB C11/(nd) RBS+HlyB C11/ (d)SoxS+ RBS+HlyB +RBS+HlyD+ RBS+TolC+T C1 1/(nd) SoxS+ RBS+HlyB +RBS+HlyD+ RBS+TolC+T C1 1

Revising constructs

Objective:

  1. Checking digestion with Eco RI of some constructs that had strange result in former electrophoresis

Samples:

  • Gel 3:
  • (d)RBS+HlyD C4/ (nd)RBS+HlyD C4/ (d)RBS+Tol C C11/ (nd)RBS+Tol C C11/ (d)RBS+QseB+RBS+QseC C10/ (nd)RBS+QseB+RBS+QseC C10/(d)RBS+SoxS C13/ (nd)RBS+SoxS C13/ (d)RBS+HlyB C10/ (nd)RBS+HlyB C10/ (d)RBS+HlyB C11/(nd) RBS+HlyB C11/ (d)SoxS+ RBS+HlyB +RBS+HlyD+ RBS+TolC+T C1 1/(nd) SoxS+ RBS+HlyB +RBS+HlyD+ RBS+TolC+T C1 1


  • Results:
Construction Total size (gene+vector) Linear vector size Gene size Result
(d)RBS+HlyD C4 3566 2070 1496 Digestion did not work. (Closed vector with same size linearized vector).
(nd)RBS+HlyD C4 3566 2070 1496 Closed vector band ok.
(d)RBS+Tol C C11 3650 2070 1580 ----
(nd)RBS+Tol C C11 3650 2070 1580 ----
(d)RBS+QseB+RBS+QseC C10 4156 2070 2086 Digestion did not work. (Closed vector with same size linearized vector).
(nd)RBS+QseB+RBS+QseC C10 4156 2070 2086 Closed vector band ok.
(d)RBS+SoxS C13 2173 2070 103 It seems that ligation is ok, fragment size band matches.
(nd)RBS+SoxS C13 2173 2070 103 Closed vector band ok.
(d)RBS+HlyB C10 4253 2070 2183 Digestion did not work. (Closed vector with same size linearized vector).
(nd)RBS+HlyB C10 4253 2070 2183 Closed vector band ok.
(d)RBS+HlyB C11 4253 2070 2183 Digestion did not work. (Closed vector with same size linearized vector).
(nd)RBS+HlyB C11 4253 2070 2183 Closed vector band ok.
(d)SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C1 1 8662 3205 5457 Digestion did not work
(nd)SoxS+ RBS+HlyB+ RBS+HlyD + RBS+TolC+T C1 1 8662 3205 5457 Closed vector band ok.



Building the devices

Electrophoresis Gel 4, 17/09/2011.
Gel 4: Ladder / pConst+RBS+SoxR+T C1 (D)/ pConst+RBS+SoxR+T C2 (D)/ pConst+RBS+SoxR+T C3 (D)/ pConst+RBS+SoxR+T C4 (D)/ pConst+RBS+SoxR+T (ND) / SoxS+RBS+IL-10+HlyA+T C1 (D)/ SoxS+RBS+IL-10+HlyA+T C2 (D)/ SoxS+RBS+IL-10+HlyA+T C3 (D)/ SoxS+RBS+IL-10+HlyA+T C4 (D)/ SoxS+RBS+IL-10+HlyA+T (ND)/ flhDC+RBS+IL-12+HlyA+T (D) / flhDC+RBS+IL-12+HlyA+T (ND)/ flhDC+RBS+GFP+HlyA+T (D)/ flhDC+RBS+GFP+HlyA+T (ND)/ SoxS+RBS+GFP+HlyA+T (D)/ SoxS+RBS+GFP+HlyA+T Test (D)/ SoxS+RBS+GFP+HlyA+T (ND) / pConst+RBS+QseB+RBS+QseC+T (D)/ pConst+RBS+QseB+RBS+QseC+T (ND)
Electrophoresis Gel 5, 17/09/2011.
Gel 5: Ladder / flhDC+RBS+HlyB+RBS+HlyD+TolC+T (D)/ flhDC+RBS+HlyB+RBS+HlyD+TolC+T (ND)/ SoxS+RBS+ HlyB+RBS+HlyD+TolC+T (D)/ SoxS+RBS+ HlyB+RBS+HlyD+TolC+T (ND)/ pSB1C3 TolC C10 (D) / pSB1C3 TolC C10 (ND)


Quantification and checking digestion of sequences to ship

Objectives:

  1. Finishing the assembly of our devices
  1. Secretion device (containing HlyB, HlyD and TolC in two manners: regulated by flhDC promoter or SoxS promoter)
  2. Th1-directed device (containing QseC and QseB sensor proteins constitutively expressed and IL-12 whose expression is regulated by flhDC promoter)
  3. Th2-directed device (containing the SoxR sensor protein constitutively expressed and IL-10 whose expression is regulated by SoxS promoter)
  1. After finishing the assembly of these parts, we aim to put together the plasmid containing the Secretion system (1.1) and the plasmid containing the sensor-response systems (the one that senses Adrenaline and responds by producing IL-12 (1.2) and the one which senses Nitric Oxide and responds by producing IL-10 (1.3).

Samples:

  • Gel 1:
  • Ladder / pConst+RBS+SoxR+T C1 (D)/ pConst+RBS+SoxR+T C2 (D)/ pConst+RBS+SoxR+T C3 (D)/ pConst+RBS+SoxR+T C4 (D)/ pConst+RBS+SoxR+T (ND) / SoxS+RBS+IL-10+HlyA+T C1 (D)/ SoxS+RBS+IL-10+HlyA+T C2 (D)/ SoxS+RBS+IL-10+HlyA+T C3 (D)/ SoxS+RBS+IL-10+HlyA+T C4 (D)/ SoxS+RBS+IL-10+HlyA+T (ND)/ flhDC+RBS+IL-12+HlyA+T (D) / flhDC+RBS+IL-12+HlyA+T (ND)/ flhDC+RBS+GFP+HlyA+T (D)/ flhDC+RBS+GFP+HlyA+T (ND)/ SoxS+RBS+GFP+HlyA+T (D)/ SoxS+RBS+GFP+HlyA+T Test (D)/ SoxS+RBS+GFP+HlyA+T (ND) / pConst+RBS+QseB+RBS+QseC+T (D)/ pConst+RBS+QseB+RBS+QseC+T (ND)
  • Gel 2:
  • Ladder / flhDC+RBS+HlyB+RBS+HlyD+TolC+T (D)/ flhDC+RBS+HlyB+RBS+HlyD+TolC+T (ND)/ SoxS+RBS+ HlyB+RBS+HlyD+TolC+T (D)/ SoxS+RBS+ HlyB+RBS+HlyD+TolC+T (ND)/ pSB1C3 TolC C10 (D) / pSB1C3 TolC C10 (ND)
  • Ladder:
  • 100-10.000 bp (Fermentas)
  • Gel Agarose concentration:
  • 1,0%


  • Results:

Construction Total size (gene+vector) Linear vector size Gene size Result
Const-RBS-SoxR-T (C1) 2753 2100 653 smaller
Const-RBS-SoxR-T (C2) 2753 2100 653 OK
Const-RBS-SoxR-T (C3) 2753 2100 653 No OK
Const-RBS-SoxR-T (C4) 2753 2100 653 OK
SoxS-RBS-IL10-HlyA-T (C1) 4170 3200 970 smaller
SoxS-RBS-IL10-HlyA-T (C2) 4170 3200 970 smaller
SoxS-RBS-IL10-HlyA-T (C3) 4170 3200 970 smaller
SoxS-RBS-IL10-HlyA-T (C4) 4170 3200 970 smaller
flhDC-RBS-IL12-HlyA-T (P2.C1) 5306 3200 2106 OK
flhDC-RBS-GFP-HlyA-T (P1.C2) 3983 2800 1183 OK
SoxS-RBS-GFP-HlyA-T (C4) 4360 3200 1160 OK
SoxS-RBS-GFP-HlyA-T (enzyme test) 4360 3200 1160 OK
Const-RBS-QseB-RBS-QseC-T (C1) 4221 2100 2121 strange
flhDC-RBS-HlyB-RBS-HlyD-RBS-TolC-T (from competent cells – C2) 8280 2800 5480 OK
SoxS-RBS-HlyB-HlyD-TolC-T (from competent cells – P1.C3) 8257 2800 5457 OK
pSB1C3-TolC (C10) 3650 2070 1580 No fragment band




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