Here we describe experiments and protocols we used in our iGEM project.
5 July 2016
Preparation of agar plates with kanamycin + chloroamphenol.
A) LB agar: requires 37 g/L of the commercial ready mix. We measured 14.8 g for 400 mL water, then autoclaved. (LB agar needs to be cool enough to touch - so not too hot - around 45-55°C)
B) Addition of antibiotics: we added the required antibiotic to the liquid agar once it was cool enough to touch. Working concentration of the antibiotic was a 1000X dilution of stock concentrations, i.e. from mg/mL to μg/mL.
C) Pouring of plates - around 25 mL of LB agar with antibiotic per plate (top tip: use pipette gun to suck up bubbles). We also prepared chemically competent cells (specifically TOP10 and BL21(DE3) E. coli strains, the former for plasmid propagation and the latter for future expression experiments).
Bacterial transformation (chemical), with Luba's plasmid DNA - to check that the cells were indeed competent, as well as their transformation efficiency. We performed a heat shock by placing microbes containing our samples at 42°C for 1 minute. Following addition of SOC medium, samples were Incubated at 37°C for 1 hour.
Transformed one of each cell type with different plasmid DNA:
- TOP10 + KT2440 enzyme in pET-29a(+)
- TOP10 + pQR801
- BL21 + KT2440 enzyme in pET-29a(+)
- BL21 + pQR801
6 July 2016
The transformations worked
7 July 2016
Abbie + Kuba + Dale plated out 4x glycerol stock BioBricks:
- 3x on chloramphenicol:
- BBa_M30109 in pSB1AC3 6/12/13
- BBa_K812014 in pAB1C3 7/26/13
- BBa_I712004 in pSB1AC3 7/26/13
- 1x on kanamycin:
- BBa_E0015 in pSB1AK3 8/7/121
8 July 2016
Abbie + Kuba + Dale checked colonies and made ampicillin plates. Unfortunately, there were no colonies from transformed BioBrick samples from 07/07/16.
- 500 / 125 / 250 mg of each antibiotic was weighed and stock solutions were prepared by adding water (to ampicillin and kanamycin) or ethanol up to 5 mL.
- 2x chloramphenicol (50 mL LB + 50 μg chloramphenicol stock solution)
- 15x ampicillin (350 mL LB + 350 μg ampicillin stock solution)
- Positive control
- BBa_1712004 in pSB1AC3
- BBa_J63008 in pSB1A3
- BBa_B0015 in pSB1AK3
- BBa_K5120 in pSB1C3
- BBa_1715004 in pSB1AC3 (chloramphenicol + control)
- BBa_J63008/BBa_J63009 in pSB1A3 (ampicillin + control)
- BBa_B0015 in pSB1AK3 (kanamycin + control)
- BBa_K5126 in pSB1C3 (chloramphenicol + control)
1. The team has made ampicillin, chloramphenicol and kanamycin stock solutions as indicated in Table 1, above.
2. We poured plates again: approx 25 mL per petri dish
3. Plating of samples:
4. Inoculation: in 50 mL falcon tubes, 5 mL of LB and 5 μL of the relevant antibiotic was added
11 July 2016
10-12:30PM : Abbie + Kuba + Dale analysed samples incubated over the weekend.
12 July 2016
13 July 2016
Today we have inoculated 4x samples to get pSB1C3, pSB1K3, pSB1A2, pSB1A3 backbones. Dale and Amandeep have minipreped 2x cultures (from the day before) in the afternoon. If today’s inoculations are successful, we hope to do more minipreps tomorrow morning
20 July 2016
Dale prepared some agar and media
8 September 2016
Today was an exciting day as we prepared our DNA for shipment for the iGEM Registry. We prepared our samples according to the iGEM protocol and dried them overnight. We also miniprepped samples containing RBS + GFP, from 2013 and 2016 distribution kits, in order to analyse them using a restriction digest. This was done for future experiments with our pH sensitive promoter GadA. We also prepared glycerol stocks for GFP samples prior to performing minipreps.
The Spy BioBrick, obtained from UCL iGEM 2010 was in the form of a glycerol stock. From the registry, it was assumed that the backbone was chloramphenicol, so an overnight culture was started.
There was no growth observed from the overnight culture, so it was decided to inoculate media containing all three antibiotics and tubes without antibiotic.
The previous days attempt to grow Spy from the glycerol stocks was unsuccessful in the three tubes containing antibiotic, along with a tube containing no antibiotic. Therefore, it was decide to transform the Spy DNA.
Transformation of Spy
The Spy DNA was transformed into BL21 competent cells. As the backbone was unknown, the transformation was plates on ampicillin, chloramphenicol and kanamycin plates, along with plates without antibiotic.
As the Spy is in ampicillin, not chloramphenicol, the 3A assembly technique was used.
Analytical Digest of Spy BioBrick
The spy transformation was successful on ampicillin, so an analytical digest was carried out to check the transformation was successful.
Preparation for Characterisation
All the reagents and consumables were prepares in anticipation of the Spy characterisation. An overnight culture was set up for the following day.
Characterisation of Spy BioBrick
The Spy BioBrick was characterised by measuring it’s fluorescence at different pHs against the fluorescence of E. Coli with a T7 promoter.