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.  

Day 8 (07/07/14): 

Today we met in the library to decide on which genes we wanted to try and insert into plasmids. We decided upon S-linalool synthase, R-linalool synthase, E-E-germacrene-B synthase, patchoulol synthase, R-limonene synthase, zingiberne synthase and geraniol synthase. We found the sequences for these and also began looking at what promoters and terminators we could use. We also began looking at using light to activate the synthesis of these genes.  

Day 9 (08/07/14): 

Today Taylor and Matt used the transformation efficiency kit to test the efficiency of the competent cells we had made. These were plated and left overnight to grow. 

We also had a meeting with Wei-Feng, and discussed the lab work we have carried out so far, the enzymes we have selected to try and put into plasmids, and the best methods to do this. Wei-Feng suggested using Gibson assembly to do this. Wei-Feng also suggested disregarding any sequences that had been used in iGem before, as we would not be creating new biobricks with them.  

Day 10 (09/07/14): 

This morning we discovered that due to an error in carrying out the transformation efficiency yesterday, cells had not grown on the plates.  

We also began designing primers for the sequences that we have chosen, and discussed using Gibson assembly as opposed to using the Biobrick method and what promoters to use.  

Day 11 (10/07/14): 

Today we narrowed down what sequences we wanted to insert into plasmids to germacrene-b synthase, patchoulol synthase, R-linalool synthase and zingiberne synthase. We started to look at removing restriction sites from these sequences and optimizing the sequences for expression in E.coli.  

Matt and Claire also carried out again the transformation efficiency kit as it had not worked yesterday.  

Day 12 (11/07/14): 

This morning we found again that the transformation efficiency had not worked as no colonies had grown on the plates.  

Ellie completed the optimization of the sequences with removal of restriction sites, and addition of prefixes and suffixes.

Day 13 (14/07/14): 

Today we repeated the transformation efficiency protocol again with cells that we knew were definitely competent, to see if the problem was with the cells we were using or with our carrying out of the protocol. These were left to incubate overnight at 37°C.  

We also carried on working on our sequences, and started looking into how much it will cost to order our sequence. As our sequences are quite long we looked into whether it will be cheaper to order the sequences in parts or as one long sequence.  

As a group we discussed ideas for the wiki, and Taylor and Alex carried on working on this. Claire designed a poster for Alex and Ellie to take to Sheffield on Friday.  

Day 14 (15/07/14): 

When we took the transformation efficiency plates out of the freezer this morning we found that once again we had no colonies. We discussed reasons for this, and prepared an agarose gel to check that the plasmid was present in the DNA samples we are using. 

We also prepared a number of agar plates and agar plates with Chloramphenicol. 

Taylor and Alex carried on working on the wiki, using the idea of a minimalistic design that we had agreed upon. We also decided that we like the logo that Rosie has designed, but that it needs some changes/improvements.  

We met with our supervisors, and discussed sequences, lab work, the wiki and future events. We decided that due to the budget we will initially only order one sequence. We decided as a team that zingiberne synthase would be the enzyme we would like to use first.   

Day 15 (16/07/14): 

We carried out agarose gel electrophoresis on the GFP plasmids we had obtained in the miniprep. However due to an error in the preparation of the gel this was unsuccessful. We also left a sample of E.coli cells in freshly prepared LB at 37°C over night to grow. 

Taylor carried on working on the Wiki, and Rosie and Alex carried on working on the logo. We also filled out the iGem safety forms and a proposal for a workshop we could hold at the YSB conference in September.  

As a group we discussed whether or not to use the enzyme patchoulol synthase as we have found out that the current production of patchouli oil is done is quite a sustainable way. We decided that using patchoulol synthase probably isn’t the best idea and to look into alternative enzymes we could use instead.  

Day 16 (17/07/14): 

Today we completed the poster and presentation for the Sheffield meet up. We also carried on working on the logo design, adding in some of the iGem colours.  

The zingiberne synthase sequence was finalised and sent to Wei-Feng to be ordered, and we design the Biobrick we will make with this sequence, including the promoter, ribosome binding site and terminator. 

In the lab Brogan and Rosie prepared some more competent cells, and also carried out agarose gel electrophoresis of the plasmid we obtained from the mini-prep, to check the plasmid was present. Unfortunately when viewed under UV, only the DNA marker was present, showing the plasmid was not present in the sample. 

Day 17 (21/07/14): 

Today we took a colony of GFP transformed cells and inoculated them in LB media. These were left to grow overnight at 37°C ready to be used in the miniprep procedure tomorrow. We also transformed the new competent cells we had made with YFP, and left these to grow overnight also at 37°C on chloramphenicol agar plates. An agarose gel was also prepared ready to carry agarose gel electrophoresis tomorrow with the plasmid we hope to obtain from the miniprep, to check the plasmid is present.  

We finalised the zingiberne synthase and alpha-farnesene sequences, so that they could be ordered and we can then begin to assemble our Biobrick.  

Day 18 (22/07/14): 

Today we carried out the miniprep of the GFP transformed cells that had been growing overnight to obtain the GFP plasmid from them. This plasmid was the run on the agarose gel we had prepared yesterday. Unfortunately whilst running this gel began to melt. We also used spectrometry to determine the concentration of plasmid we had obtained, and we had a high concentration. 

We also sent emails to five different local and national newspapers to begin trying to spread the word about synthetic biology. We also emailed an iGem team from Paris and Sheffield iGem about collaborations.  

We also met with our supervisors and discussed the ordering of sequences, Boston trip, outreach and other iGem events. 

Ellie, Claire and Lisa developed sequences for R-linalool synthase and Patchoulol synthase.  

Day 19 (23/07/14): 

Today our sequence for zingiberne synthase was ordered. It will hopefully arrive in eight days. We confirmed the sequences for patchoulol synthase and R-linalool synthase, and found parts in the repository that we need to order.  

Taylor carried on developing the wiki, creating a ‘landing page’ that people will see when they look at our site until it is completed. 

Brogan and Rosie found quotes for hotels and flights for Boston, and also emailed newspapers and planned outreach. 

Day 20 (24/07/14): 

This morning we decided upon a constitutive promoter, a ribosome binding site and a terminator that we wanted to transform. This is so we will have these ready when our sequence for zingiberne synthase arrives. We also decided to transform limonene synthase (from the iGem kit), so we can practice doing the restriction digests and ligations.  

We prepared chloramphenicol of the correct concentration, and then prepared agar plates with chloramphenicol to grow our transformed cells on.  

We then carried out the four transformations, using parts BBa_J23108 (promoter), BBa_J1742111 (limonene synthase), BBa_B0015 (terminator) and BBa_B0030 (RBS) from the plates in our kit. We  plated each transformation twice and left to incubate overnight at 37°C. 

Day 21 (25/07/14): 

This morning we counted the plates of transformed cells that we had been growing overnight. 

BBa_J23108: 0 colonies, 0 colonies 

BBa_B0030: 0 colonies, 1 colony 

BBa_J1742111: 8 colonies, 7 colonies 

BBa_B0015: 2 colonies, 8 colonies 

As there was no real growth for the promoter or RBS we will retransform these next week. We will miniprep the limonene synthase and terminator transformations next week as well.

Day 22 (28/07/2014): 

Today we found a new constitutive promoter and ribosome binding site to transform. These are parts BBa_J23109 (promoter) and BBa_B0032 (RBS). These were transformed, plated, and left to grow overnight at 37°C.  

We selected a colony of the E.coli transformed with part BBa_1742111 (limonene synthase) and part BBa_B0015 (terminator) and inoculated these in LB. These were also left at 37°C overnight to grow ready to carry out the miniprep tomorrow. 

Day 23 (29/07/14): 

Today we found that colonies had grown on the plates transformed with part BBa_B0032 (RBS) yesterday, but no colonies on the plates transformed with part BBa_J23109.  

A colony of BBa_B0032 (RBS) transformed E.coli cells was inoculated in LB media and left to grow overnight at 37°C. 

We selected another new constitutive promoter, part BBa_J23119 and carried out the transformation of this, as well as retransforming part BBa_J23109. These were both plated and left at 37°C overnight to grow. 

We carried out a miniprep of part BBa_1742111 (limonene synthase) transformed cells and part BBa_B0015 (terminator) transformed cells that had been grown up overnight. The plasmid obtained from these was stored at -20°C. 

We also had a meeting with Wei-Feng and Luca, in which we discussed the planning of Boston and YSB 2.0, our ordered sequence and general lab work.  

We also skyped Paris Bettencourt and discussed a possible collaboration between the teams. 

Day 24 (30/07/14): 

We had a lot of growth of both promoters, parts BBa_J23109 and BBa_J23119, however we realised this is as they had been grown on plates with no chloramphenicol and therefore transformed colonies had not been selected for. We therefore transformed both of these parts again, plated these and left them to grow overnight at 37°C.  

We had been growing up overnight E.coli cells transformed with part BBa_B0032 (RBS). We carried out a miniprep of these and collected the plasmid.  

Plasmid obtained from the minipreps of the E.coli cells transformed with parts BBa_1742111 (limonene synthase), BBa_B0015 (terminator) and BBa_B0032 (RBS), was then ran on an agarose gel to check if the plasmid is present. All three plasmids were present.  

Day 25 (31/07/14): 

Colonies of E.coli transformed with parts BBa_J23109 and BBa_J23119 had both grown overnight. Colonies were selected and inoculated into 5ml of LB to grow at 37°C overnight. 

We performed a single restriction digest of limonene synthase plasmid using the enzyme EcoR1, another single restriction digest using the enzyme Pst1, and a double restriction digest using both enzymes. The fragments obtained from these digests were then run on an agarose gel. However, these digests did not work. 

Day 26 (01/07/14): 

E.coli transformed with the promoter parts BBa_J23109 and BBa_J23119 that had been grown up overnight were minipreped, and the plasmid obtained from each stored at -20°C.  

As yesterday’s digest of limonene synthase had not gone correctly, this was repeated today. Limonene synthase gene in the plasmid backbone psb1C3 was digested singly with EcoR1 and Pst1, and doubly with both enzymes. This was then run on the gel again and the correct parts had been obtained, although there had been a problem with the gel so this will be repeated on Monday.  

Taylor and Alex have also carried on designing and coding the wiki. 

Day 27 (04/08/14): 

We carried out a restriction digest using just EcoR1, just Pst1 and using both enzymes on parts BBa_J23109 (promoter), BBa_J23119 (promoter), BBa_B0032 (RBS), BBa_174211 (limonene synthase), and BBa_B0015 (terminator).  

We than ran the digests on agarose gels to check the correct size parts had been obtained. Due to running out of time the gels were not able to be left running long enough and therefore when imaged the bands had not separated. We will therefore repeat this work tomorrow. 

Day 28 (05/08/14): 

We carried out a restriction digest using just EcoR1, just Pst1 and using both enzymes on parts BBa_J23109 (promoter), BBa_J23119 (promoter), BBa_B0032 (RBS), BBa_174211 (limonene synthase), and BBa_B0015 (terminator).  

We then ran the digests on agarose gels to check the correct size parts had been obtained. These all showed the expected bands so the restriction digest had worked.  

We also had a meeting with our supervisors in which we discussed upcoming deadlines, London YSB 2.0 planning, Boston travel, outreach and collaborations and lab work.  

Taylor carried on working on the wiki, creating the template design that the information will then be loaded onto. 

Day 29 (06/08/14): 

Today we prepared two agarose gels that we can run a digested limonene synthase plasmid on tomorrow. 

We carried out a transformation of parts BBa_174211 (limonene synthase) and BBa_B0015 (terminator), into competent E.coli cells. We also revitalised our colonies BBa_J23109 (promoter), BBa_J23119 (promoter) and BBa_B0032 (RBS) transformed cells be replating them.  

We also compiled an inventory of all of the cells, plasmids, enzymes etc. that we have in stock, so that we can make/order more before we run out.  

Taylor also uploaded our wiki template so we can start to upload information onto it. 

A double restriction digest of limonene synthase plasmids was carried out using the enzymes EcoR1 and Pst1. These were left to incubate overnight at room temperature. 

Day 30 (07/08/14): 

The digested limonene synthase plasmid was ran on two agarose gels to separate the psb1C3 backbone from the limonene synthase part. The bands were then visualised and cut out of the gel. A DNA gel extraction kit was used to extract the DNA from the gel. This DNA was then stored at -20°C. 

We inoculated cells transformed yesterday with parts  BBa_J23109 (promoter), BBa_J23119 (promoter), BBa_B0032 (RBS), BBa_174211 (limonene synthase) and BBa_B0015 (terminator) in LB and left overnight at 37°C.