"
Team:Cambridge/Voltage/Lab Work
From 2008.igem.org
15th July
Conference in Edinburgh
Meet with the other UK iGEM teams.
16th July
Conference in Edinburgh
Presentation of projects to the other teams and to the attendees of the SynBioStandards network meeting.
17th July
Progress
-Ordered mutant E.coli chassis to test: Kch-, KdpD-, KdpE-, KdpF-E-
-Designed two plasmids:
-Sourced V.harveyi BB170 culture to obtain glutamate-gated potassium channel gene
-Modelled protein structure
-Designed primers for extraction of Kdp Operon from E.coli and Glu Gated channel gene from V.harveyi
-Decided on oxygen electrode for small voltage change measurements
-Extracted BioBricks from registry to use in plasmids:
- Promoter BBa_J23100
- RBS BBa_B0030
- Terminators BBa_B1002 and BBa_B1006
18th July
Wet Work
There was no growth on the plates spread yesterday. There has been a few adjustments to the protocol.
Biobrick Extraction
| Class | Registry | Part Kit Location | Tube Label |
|---|---|---|---|
| Promoter | BBa_J23100 | 2008 1002-10E | F |
| RBS | BBa_B0030 | 2008 1002-5G | G |
| Stop | BBa_B1002 | 2008 1016-8A | H |
| Stop | BBa_B1006 | 2008 1016-8D | I |
| Promoter | BBa_J23113 | 2008 1002-12B | J |
| Promoter | BBa_J23114 | 2008 1002-12C | K |
| Control | pUC19 | L |
Bigger, better, faster, stronger
We found we could not extract enough DNA from the registry, so we are upgrading the extraction to the "bigger, better, faster, stronger" method.
Warm 50μL of EB in Eppendorf tubes at 50°C and add 4 punched spots. Keep it warming at 50°C for 20mins and spin down for 3 minutes at 15,000 g. Warm agin for 10mins and spin down again for 3mins. Pipette off the liquid which should have the DNA in. We then confirmed with PCR.
Each sample except the control was then put through 34 cycles of PCR.
The following were added to eppendorf tubes:
5μL of DNA in EB buffer
2.5μL of each primer for Biobrick vectors
25μL of Finnzymes mastermix
15μL of sterile distilled water
And all 50μL were then run through the PCR reaction and 17μL of product run on an E-Gel with 3μL dye.
The results gave large amounts of DNA in each lane for all Biobricks except F. The original extracts of G,H,I,J,K and L were then used for the following transformations.
To chilled tubes add 5μL of DNA + EB solution to 5μL of Cells ( Chemically competent Top 10 )
Ice for 30 minutes Heat shock at 42°C for 60 seconds Ice for 2 minutes Add 500μL of SOC Incubate at 37°C overnight
Prepare Agar plates : Add 200μL of Amp (100mg/mL) into 200mL of LA ( Final conc. of 100μg/mL ) and add 100μL of Amp (100mg/mL) into 200mL of LA ( Final conc. of 50μg/mL ) Plate neat samples of G,H,I,J,K,L onto both types of plate and incubate overnight.
21st July
Wet Work
Aims for Today
The results of yesterday's modified protocol still yielded no growth on the plates. Using yesterday's PCR product our aims were:
- Cut promoters with spe1
- Cut RBS with Xba1
- Ligate these together and perform further PCR amplification of linear fragments.
- Cut first 'stop' with spe1
- Cut second 'stop with xba1
- Ligate these together and perform further PCR amplification of linear fragments.
- Finally, put all the above elements into a vector plasmid.
- End of day: Aims completed
Length
- Promoter = 35 bases
- RBS = 16 bases
- Stop (B1002) = 35 bases and Stop (B1006) = 39 bases
22nd July
Wet Work
- Inoculated 10mL LB with E.coli strain MG1655
- Inoculated 10mL LB with E.coli strain TOP10 transformed with BioBrick J5311
16 LA plates were poured with 8 having 25μg/mL Kanamycin, ready for mutant strains to be grown on.
The gel extracts from the ligation of primer and rbs were extracted using Zymoclean gel extraction kit. Hopefully these now contain linear DNA of BBa_J23113 and BBa_B0030, and BBa_J23114 and BBa_B0030.
BioBricks J45996, J45995 and J45250 were also extracted from the registry, using the "bigger, better, faster, stronger, method", in order to provide template to extracted the OsmY promoter.
23rd July
Wet Work
- Recovered Biobrick plasmid YFP from previous culture.
- 1.6 ml of cells according to Zippy Plasmid Miniprep Kit protocol (60μL Elution Buffer used)
- Added 20ml LB to 10ml innoculation of E Coli MG1655 (see 22/07/08) to increase volume in preparation for experiments tomorrow on K+ tolerance in media.
