Day 1 (25/06/14): 

After a meeting with our advisors yesterday, we came into the lab this morning and began learning how to prepare competent cells.  

We prepared: 

100mM CaCl2 – autoclaved 

100mM CaCl2 + 30% v/v glycerol – autoclaved 

100ml LB media (1g Tryptone, 0.5g Yeast extract, 1g Sodium Chloride) - autoclaved 

E.coli was added to 5ml of LB and incubated overnight at 37° 

Pipette tips, conical flasks, bottles etc were also autoclaved ready for tomorrow 

Also discussed possible project ideas and booked a room in the library to discuss theses as a group tomorrow.  

Day 2 (26/06/14): 

Today we carried on with the preparation of the competent cells. 

E.coli that had been incubating overnight was inoculated into LB and grown at 37°C until the OD600 reached 0.4. Cells were then cooled in the cold room and then centrifuged at 3000rpm, 4°C for 8mins. Pellet was resuspended in 100mM CaCl2, incubated on ice and centrifuged again. Pellet was again resuspended in 100mM CaCl2, 30% v/v glycerol and divided into 33 x 100µl in eppendorfs and stored at -80°C.  

We began to prepare 100ml of LB agar (1g Tryptone, 0.5g Yeast extract, 1g Sodium Chloride, 2g agar), this was autoclaved but we then discovered we had no Chloramphenicol so could not continue preparing the plates.  

We also spent a few hours discussing parts and looking through the registry, and discussing different ideas. 

Day 3 (27/06/14): 

Today we carried out the transformation of our competent E.coli cells.  

Firstly we prepared 500ml of LB agar (5g Tryptone, 2.5g Yeast extract, 5g Sodium chloride, 10g agar) and autoclaved. Once this had cooled we added 0.5ml (1:1000 dilution) of chloramphenicol and made the plates. These were then allowed to set.  

We then carried out the transformation by thawing four eppendorfs of our competent cells on ice. 2µl of resuspended DNA (GFP open reading frame), was added to the competent cells, and 1µl of RFP control was added to the control competent cells. Cells they were incubated on ice for 30mins and were then heat shocked in a 42°C water bath for 60s and place back on ice for 5mins. 1ml of LB media was added to the cells and they were then incubated at 37°C for 2 hours. 50 and 100µl of the cells were plated on the LB agar with chloramphenicol we had prepared and left to grow at room temperature over the weekend. 

Day 4 (30/06/14): 

Over the weekend our plates had been growing, both plates of GFP transformed cells showed many colonies of E.coli. The two control plates both only had a few colonies and their morphology was very different to the colonies on the other plates. We therefore repeated the control plates.  

We picked a single colony from each plate we had grown and inoculated them in glycerol stock and left them overnight to grow ready to carry out the miniprep tomorrow.  

Day 5 (01/07/14): 

This morning we centrifuged our inoculated cells but no pellet was produced. We found out this is because they should not have been inoculated in glycerol stock. We therefore took two more colonies from the 100µl plates and inoculated them in LB and left to grow overnight.  

As we had no growth on our repeated control plates we also repeated the transformation, using RFP for the controls and YFP for the non-controls. These were left to incubate overnight at 37°C.  

In the afternoon we had a meeting with Mark Shepherd, to discuss ideas. By the end of the meeting we had decided to further research into the production of esters in E.coli, but also to carry on researching into BMCs.  

Day 6 (02/07/14): 

We took two colonies from the 100µl plates of cells containing YFP and inoculated them in LB media. These were left to grow overnight.  

We centrifuged the cells transformed with GFP that had been inoculated in LB media overnight. Using the pellet of cells produced we followed the miniprep protocol to obtain the plasmid DNA.  

Day 7 (03/07/14): 

This morning we met to discuss ideas for the project. After the meeting with Mark Shepherd we had spent the last few days researching the idea of targeting antimicrobial peptides to BMCs to deliver them to the gut and for treatment of gastroenteritis/other conditions, or alternatively using E.coli to produce fragrances. We discussed the pros and cons of each project and collectively decided to further research into using the production of fragrances in E.coli as an environmentally friendly alternative to their current industrial production.