Team:TUDelft/3 August 2009

From 2009.igem.org

Lab Notebook

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3 August 2009

Tim Weenink

I stole p(tetR) promotor and double terminator from the delay team to redo the !B and !C assemblies. The ligation products were run on Calin's gel (see below). The transformants were plated on Cam agar plates (unfortunatele only LB agar was left that seemed infected.) and stored in the 37 degrees C stove.

Sriram

All the transformations done on friday gave growth. However comparing to the chemically competent cells prepared by TMF buffer (RbCl) the number of colonies from TSS buffer competent cells was less. However the concentration of DNA added plays a major role in the efficiency of transformation. The λp-R-GFP-T DNA isolated by miniprep had a concentration of 116.5 ng/µl which gave huge number of colonies compared to the diluted pSB1K3 with mRFP biobrick. The electro-transformation of λp-rep biobrick was successful and gave good colonies. hence we have decided to work with electro-transformation hereafter.

Thus we can very well start the assemblies today. We did restriction for the DNA samples needed for 8 assemblies. We might have some problem with backbones but only in future assemblies.

Gel03082009-delay.png
Well No. Sample Name Expected Plasmid Size Status
1 pTet-Upstream
2 RBS-cI-RBS-Downstream
3 pSB1C3-plasmid
4 RFP-Upstream
5 Term-Downstream
6 pSB1C3-plasmid
7 pLacI-Upstream
8 RBS-Downstream
9 pSB1C3 plasmid
10 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]
11 TetR-Upstream
12 Term-Downstream
13 pSB1C3 plasmid
14 pTet-Upstream
15 Lock3C-Downstream
16 pSB1C3 plasmid
17 cI-Upstream
18 Term-Downstream
19 pSB1C3 plasmid
20 pLambdain-Upstream
21 RBS-GFT-T-Downstream
22 pSB1C3 plasmid
23 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]

Calin

Tim pointed out that the plasmids should be linearized before doing the gel. Here are possible digestions on some of the parts:

Backbone Part Unique Sites Present
pSB1A2 oriT-R AatII AccI AflIII BglI BspLU11I BstAPI
DrdI Eam1105I EcoRI FspI NspI PstI
PvuI RsaI ScaI
SgrAI SpeI SspI
StyI TatI VspI XbaI XmnI ZraI
BBa_J61002 strong promoter AatII AlwNI AvrII BglI BsiWI BspLU11I
BstXI DrdI EcoRI FspI HaeII HpaI
NcoI NspI PflMI PshAI PstI PvuI
PvuII RsrII ScaI SgrAI SpeI SspI
TatI VspI XbaI XmnI ZraI
pSB1AK3 ccdB AatII AccB1I AccI BamHI BanII BglI
BspLU11I
BsrGI BssHII BstXI BstZ17I BtrI
ClaI DraIII DrdI Eam1105I EcoNI EcoRI
FspI HaeII
HincII HindIII MscI
NruI NspI PflMI PstI ScaI SgfI
SpeI
SrfI XbaI XmnI ZraI
pSB4C5 ccdB AatII AccI AclI AflII ApaLI BamHI
BbeI BseRI
BsrGI BssHII BstAPI BstXI
BstZ17I BtrI Cfr10I EcoRI EcoRV HaeII
HincII KasI NarI NdeI PstI
PvuII SacI ScaI SfoI
SmaI SpeI
SphI
SrfI StyI XbaI XmaI ZraI

Growth on all plates. The pSB4C5 and R751 plates were parafilmed and placed in fridge.


Did linearization of oriT-R, promoter, pSB1AK3, and pSB4C5 using EcoRI.

Did digests of oriT-R (9uL) and pSB4C5 (4.5uL).

Did ligation CA and CB:

Assembly name position Components volume in µl
CA Upstream pTet-GFP 2
Downstream oriT-R 2
Backbone pSB4C5 2
H20 11
CB Upstream rbs-GFP-term 2
Downstream oriT-R 2
Backbone pSB1C3 2
H20 11


Ran a 2% gel to check various parts and digests.

Gel03082009-conj.png
Well Part Expected Plasmid Size Status
1 oriT-R linear 2079
2 oriT-R old digest 2079 + ~278
3 oriT-R control 2079
4 oriT-R new digest 2079 + ~278
5 promo linear 2983
6 promo control 2983
7 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]
8 pSB1AK3 linear 3864
9 pSB1AK3 control 3864
10 pSB4C5 linear 3896
11 pSB4C5 control 3896
12 pTet digest 2079 + 937
13 GFP-gen digest 2079 + 878
14 pSB1C3 digest 2072 + 675
15 [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder]
16  !B assembly ligation circular  ?
17  !C assembly ligation circular  ?

Orr

Made some LB Agar medium and some agarose gel with pre-made TBE at concentration 1x.