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The Big Birkbeck House

Notebook

Week 1 in the Big Birkbeck House

A joint collaboration between the UCL, Biohackspace & Birkbeck iGEM teams were held in order to train team members in certain roles within each team. The organised boot camp took place over a week. Teams all met in mornings to carry out basic lab training. In afternoons, 3 specific training routes were followed; DIYbio, Software/Automation & extra lab. Members from each formed afternoon groups.

Monday 15^th June

Morning Lab

Monday morning consisted of a Lab safety induction & a discussion on the standardised cloning strategy to be applied throughout the iGEM competition. The common major feature of each plasmid backbone has a prefix (to the 5' end of the biobrick) & suffix (3' end of the biobrick). The prefix and suffix restriction sites are highlighted in Figure 1. The standardised engineering procedure works by cutting the vector backbone in the suffix region while inserts (if already cloned into a vector) are cut in the prefix and suffix (cartoon representation of reactions are displayed in Figure 2). The ligation of insert and vector backbone will yield a recombinant plasmid with a scar as the XbaI & SpeI sites anneal.

Figure 1: Prefix & Suffix Restriction Sites.

Figure 2: Cloning Strategy.

This interlab study aims at using the same protocol in expression of rfp in E. coli cells. With reference to Figure 3, the 2006 Berkeley iGEM team characterised each of the promoters. iGEM teams across the globe will quantitatively measure the fluorescence of RFP & GFP with respect to each promoter listed. This will generate a large data set and therefore a statistically more reliable conclusion of the original experiments.

On day 1, the main goal in the morning lab was to make a ligation reaction ready for transformation on day 2. The backbone (pSB1C3 based) had to be cut with SpeI & PstI which linearised the vector backbone by cutting into the suffix(as highlighted in Figures 1 & Figures 2). The insert dervived from pSB1A2 by a double restriction digest using XbaI & PstI. In order to verify reactions had worked, each of the double digests were ran on a 1% (wt/vol) agarose gel (100 V for 1 hr), visualised by ethidium bromide staining and visualising bands using a U.V. light source.

After reaction verification, a ligation was performed by using ~75 ng of insert to 25 ng of vector. The reaction was carried out at room temperature for 30 minutes and the heat killed at 80°C for 20 mins. Reactions were stored at 4°C overnight for transformation.

Meet & Greets

All team members were invited to the anatomy building of UCL to meet each other. After a brief 30 minutes of introductions, A skype talk was held with Randy Rettberg (the president of the iGEM foundation). The talk consisted of Randy Rettbergs general background, interest in synthetic biology & the origins of the iGEM competition. A Q & A session was held after the talk.

A previous iGEM team member (UCL 2014, Georgia Bondy) held an interactive talk on the division of labour within groups. Emphasis was placed on the need for team members to take responsibility for one aspect of the project. The "babies" had to be nurtured by the team member who chooses to be responsible for each task.

Tuesday 16^th June

Morning Lab

On tuesday morning, groups were divided into sub-groups with the different subgroups using different methods in transformation. For electroporation, the ligation reaction had to be de-salted prior to electroporation. The chemical transformation protocol did not require this step and 2 μL of ligation reaction was pipetted straight into the compotent cells. E. coli cells were out grown in SOC media for 1 hour before plating on LB media containing chloramphenicol (20 μg/mL) to select for the transformants. A negative control of cells treated with 2 μL of distilled water was carried out.

Software

The first talk in the software route was on "automated synthetic biology" by Chris Grant. The main issues in this talk were the streamlining of high-throughput processes by the use of robotics. The next talks were on computer modelling of metabolic pathways/genetic circuits (by Miriam Leon & Rob Stanley). An interactive workshop took place after the talk with the repressilator system being used as an example.

DIY Bio

The overall aim of this set of workshops was to construct an open souce spectrophotometer. Software for data collection/manipulation was downloaded. The basic principles of 3D printing was explained. Components of the open source we constructed using 3D printing.

Extra Lab

In the extra lab sessions, the tricks of the synthetic biology trade were put to use. The use of molecular crowding agents (namely PEG) was used in order to spead up the ligation reactions. The use of PEG reduced the reaction times by 50%!

Wednesday 17^th June

Morning Lab

The plates that were inoculated on Tuesday were checked for any colonies. no growth was observed on the negative control plate. The chloramphenicol provided the selective pressure that would promote the growth of E. coli cells that were transformed with plasmid. Single colonies were picked and grown in liquid media (5 mL LB containing 20 μg/mL chloramphenicol) for plasmid harvesting. It was noted that there was significantly more transformants in the electroporation method of transformation. It was difficult to pick single colonies off the electroporation plates.

Software

The principles of wiki design was discussed by Lewis Moffat (UCL 2014 iGEM team). The logic behind html coding was explained. As a workshop a spoof page was set up and images & text were uploaded to the page. Everyone involved in the workshop got hands-on experience in directly uploading & editing a wiki page.

DIY Bio

A tour of London Biohackspace was given to people involved in this group. A talk on DIYbio was given by Ilya Levantis. The parts previously constructed for the open source spectrophotometer were put together.

Extra Lab

Plates that were inoculated on Tuesday were checked for any transformants. The practical was followed by a talk on advanced assembly techniques (by Dr. Vitor Pinheiro).

Thursday 18^th June

Morning Lab

Overnight cultures of the transformants were ready to harvest! A glycerol cell stock of E. coli transformants was made by harvesting 0.5 mL of cells and mixing with 0.5 mL of 50% (vol/vol) sterile glycerole (25% final [glycerol]). 1 mL of culture was lysed and used to harvest plasmid DNA.

Purified plasmids were screened for the presence of the insert by a diagnostic restriction digest. This restriction digest effectively liberates the insert from the vector and should yield 2 clear bands. EcoRI & PstI were used to liberate the insert (note that where ligation occurs between XbaI & SpeI in the suffix forms a scar, therefore the EcoRI restriction site in the suffix must be used as a diagnostic - refer to Figure 1). Agarose gels were ran of the digests. Varying degrees of success was observed.

Software & Extra Lab

The extra lab & Software groups had a skype interview with Jacob Beal about the interlab study. He laid out the main aims of the interlab study and answered questions that teams members had over the project.

DIY Bio

The open source spectrophotomoeter was tested. A strain of E. coli expressing rfp from a plasmid was used to test the spectrophotometer.

Friday 19^th June (THE MINI JAMBOREE!)

The time had came to showcase the skills which we had learned over the week. The iGEM teams met up in the morning in order to prepare for the presentations in the afternoon. Each of the iGEM teams had to also discuss their biobrick of choice related to their project. Each presentation lasted 5-10 minutes.

The teams that were split into different afternoon workshops also had to present what they have learned over the course of the week. Each teams presentation lasted 5-10 minutes (with exception to the software team lasting ~3 minutes - short & sweet!).