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Reporter: Week 8 July 3-July 9
From 2011.igem.org
Contents |
Sunday, July 3
Assembly of Fusion Parts, Day 5
All of the assemblies from 7/2 and the promoter+RBS assembly (J23100+B0034) were verified using agarose gel electrophoresis. Since the ladder was contaminated, the relative sizes of the assemblies were analyzed. This analysis showed that the J23100+B0034 was much smaller than the other assemblies, the XylE+both linkers were just under 1000 base pairs, and the GFP+cleavage sites were a little smaller than the XylE+linker parts. Thus, each construct was grown in culture overnight in order to extract plasmids for sequencing.
Plasmids from the four cultures made on 7/1 were extracted using the Omega Bio-Tek miniprep protocol. These plasmids were stored in the 4°C fridge until they could be sent to sequencing on 7/5.
Monday, July 4
Assembly of Fusion Parts, Day 6
Plasmids were extracted from the eight cultures made on 7/3 using the Omega Bio-Tek miniprep protocol. Since the sequencing center is closed for the holiday, these plasmids will be sent to sequencing tomorrow, along with the plasmids extracted on 7/2.
Tuesday, July 5
The following plasmids were sent to sequencing:
J23100+B0034 (A and B)
LacZ+Imp linker
LacZ+Small linker
LacZ+10 AA linker
XylE+Imp linker
XylE+Small linker
XylE+10AA linker
GFP+cI cleavage site (A and B)
GFP+tev cleavage site (A and B)
Results: The J23100+B0034 (colony A) sequence results showed that the assembly worked, so now we have a promoter in front of a ribosome binding site. The cells from this colony were used for freezer stock. The rest of the plasmids were vector background and were discarded.
The linkers were inserted behind XylE and the cleavage sites were inserted behind GFP.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Inserts using PstI and NgoMIV: | small linker Imp linker 10AA linker cI cleavage site tev cleavage site |
| Vectors using PstI and AgeI: | GFP XylE | |
| Ligation | XylE+small linker XylE+Imp linker XylE+10AA linker GFP+cI cleavage site XylE+tev cleavage site | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated on kanamycin resistant plates. | |
