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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. | |
Wednesday, July 6
Prepared M9 media following this protocol.
The assemblies from Tuesday, July 5 were verified through gel electrophoresis. The gel showed bands all slightly above 1 kbp, so two colonies of each of the constructs were grown in culture overnight.
Thursday, July 7
The following plasmids were extracted for sequencing:
GFP+tev cleavage site (A and B)
GFP+cI cleavage site (A and B)
XylE+small linker(A and B)
XylE+Imp linker (A and B)
XylE+10AA linker (A and B)
Results: The GFP+tev cleavage site, GFP+cI cleavage site, XylE+small linker, XylE_Imp linker and XylE+10AA linker all looked good, so the cells containing these plasmids were used for freezer stock.
The promoter+RBS (J23100+B0034) construct placed in front of GFP+cleavage sites and XylE+linkers.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Insert PCR | J23100+B0034 | |
| Restriction Digest | Insert using EcoRI and SpeI: | J23100+B0034 |
| Vectors using EcoRI and XbaI: | GFP+tev cleavage site GFP+cI cleavage site XylE+small linker XylE+Imp linker XylE+10AA linker | |
| Ligation | J23100+B0034+GFP+tev cleavage site J23100+B0034+GFP+cI cleavage site J23100+B0034+XylE+small linker J23100+B0034+XylE+Imp linker J23100+B0034+XylE+10AA linker | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. | |
The tev+K3 that had one base pair changed (cloned on June 10) was mutated using the Richard Lab double primer site directed mutagenesis protocol. The extension time was 1:10.
Friday, July 8
The cI cleavage site was amplified through insert PCR. Also, more GFP+K3 was grown up so more plasmid could be extracted. Both of these steps were done so in order to perform the cleavage site+GFP assemblies tomorrow.
The additions of the promoter and RBS (J23100+B0034) to the five parts from July 7 were verified through colony PCR (extension time=1:45) and gel electrophoresis. The gel showed that colonies of J23100+B0034+GFP+tev cleavage site, J23100+B0034+CylE+Imp linker, and J23100+B0034+XylE+10AA linker contained plasmids of the correct size. Cultures of these colonies will be grown up closer to Monday, when the plasmids can be sequenced.
Saturday, July 9
No colonies grew from the tev protease+K3 mutagenesis from 7/8. Thus, the double primer method was repeated with an extension time of 1:42.
The cleavage sites were placed in front of GFP.
| Protocol | Part Involved with Protocol | |
|---|---|---|
| Restriction Digest | Inserts using EcoRI and AgeI (buffer 1): | tev cleavage site cI cleavage site |
| Vector using EcoRI and NgoMIV: | GFP | |
| Ligation | tev cleavage site+GFP cI cleavage site+GFP | |
| Transformation/Plating | The above ligations were transformed into Escherichia coli cells and plated onto kanamycin resistant plates. | |
