• Training
• Wiki

Training

We started a discussion of our sub-projects, doing some research into project presentation including: wiki layout, t-shirt design and possible team logos. We looked at precedents for inspirational ideas.,

We then learnt about Computational Modelling and BioBrick concepts. Decided to start with biophysical modelling of the lipid membrane of Bacillus subtilis and modelling of lipid synthetic pathway. Later in the day, the entire team met to discuss aims and progress of our work.

Wiki

Initial designs for the wiki have been created and uploaded. Currently all style sheets and javascript pages are stored externally until the design has been ratified by the team. Front-page image carousel has been created and initialised with blank colours to demonstrate what it shall look like until actual photographs have been taken and edited.

• Modelling Training
• Wiki

Modelling Training

We started off working on a Modelling Exercise to identify components and species of the subtilin biosynthesis pathway. We also sketched down some potential logo ideas and created a shortlist of favorites.

We then went on to learn about the differences and similarities between stochastic and deterministic modeling. We also used rule based modeling such as RuleBender and BioNetGen. Using the Michaelis-Menten kinetics model as an example, we alter the parameters and rates of reaction to observe the effects.

Wiki

The wiki design was agreed upon and further developed. A front-page image display has been created, developed and set up with dummy text and placeholder images. Discussions have been held over the layout for the calendar, the general consensus being that as few a clicks as possible should be needed to switch between and overall summary of what was done and the more detailed view. Designs are started with the idea being to create a large calendar holding daily headlines that spawn a non-obtrusive pop-up window containing more detail.

• Modelling Training

Continued learning about stochastic and deterministic modeling and looking at converting the rate parameters in RuleBender. Work was also still ongoing with the logo.

• Basic Lab Skills

Basic Lab Skills

On our first day in the lab, we were briefed on various lab health and safety policies to ensure we maintained a safe working environment. We were then led by our supervisors on a tour around the lab to inform us about the layout. Once we were familiarised with everything, we officially started our first lab session.

We started by callibrating our pipettes and learning how to operate them, then we were shown how to innoculate bacteria to a liquid culture. After this we prepared bacteria streak plates.

In the afternoon, we extracted GFP and RFP bricks from the iGEM 2013 biobrick plate and transformed it into competent Escherichia coli cells. These procedures were performed using the following protocols ([PROTOCOLS DESPERATELY NEED ADDING]). We then plated those cells onto LB with chloramphenicol selection marker plates.

• Basic Lab Skills
• Wiki

Basic Lab Skills

When we first arrived in the lab, we checked on our plates to see if anything grew. The RFP plates showed a lot of collonies, while the GFP plates did not work as expected. The next thing we did was too view the plates under the fluorescent inverted microscope.

We had an introductory lecture on basic molecular biology and biobrick design.

Wiki

The calendar has now been created, with the emphasis on both user interaction (simple and obvious to use) as well as ease of editing (the members of the team not familiar with HTML and JQuery should easily be able and add to the calendar). A rough JQuery script has been written to convert standard wiki MediaWiki code into the format used by the Calendar. This will need extensive debugging before the Calendar can be put into use.

• Wiki

Wiki

The Calendar was fully debugged and deployed over the weekend whilst no one was in the lab. This means that as of tomorrow the entire team will be able to edit and add to the calendar with ease. A brief document has also been written that will provide details on how to edit some of the more complex sections of the wiki. This document will be added to as more and more functionality is added into the wiki.

• Basic Lab Skills
• BioGame
• HBsu BioBrick clonning strategy

Basic Lab Skills

The first thing we did was to transfer collonies of bacteria into LB. Then we extracted the plasmids via (plasmid prep) protocol, and check the concentration of plasmids through the use of nanodrop spectrophotometry. Next,we prepared 1% Agarose and whilst waiting for it to set we performed a single digest with (Xbal or Pstl) and double digest with both restriction digest. Finally, we load our samples to the gel in addition with DNA ladder and control.

BioGame

Work began on the BioGame. Largely contained to simply choosing the best method of implementation for the game. Considered where:

• Android SDK (with no game engine/plug ins)
• LibGDX
• OpenGL
• LWJGL

Ultimately the choice was made to use LibGDX due to the documentation available on it, the ease at which games can be developed and the consistent structure it provides with standard gaming development. After this decision was made, a series of tutorials were found and followed to provide a base of knowledge going into developing the application itself.

HBsu BioBrick Cloning Strategy

The team has been looking in literature for a protein that binds non-specifically to DNA. This will aid the visualization of Genome Shuffling among L-forms by enabling the team to see sections of DNA that have come from two different Bacteria. The decision was made to use the HBsu protein which is encoded by the hbs gene in B.subtilis because it is well characterised and should remain associated with the DNA for a long period of time. The HBsu will be conjugated with a Florescent Protein such as GFP and RFP.

• Wiki work
• HBsu BioBrick Cloning Strategy

Wiki work

We came up with a script, story board and a list of materials needed for the introductory video on our wiki. We had fun learning to crochet in order to create characters for the video. Furthermore, we updated the team profile page on the wiki by adding individual pictures of all team members.

HBsu BioBrick Cloning Strategy

An initial design for the BioBrick has been created. It includes Kanamycin resistance cassette (BBa_P1003), a lacI Promoter under a strong RBS (BBa_B0034), the BioBrick and a double terminator. Golden Braid assembly is planned to be used to create the HBsu BioBrick.

• Gus Reporter BioBrick
• BioGame
• HBsu BioBrick Cloning Strategy

Gus Reporter BioBrick

   ~~ DETAILS OF THE INITIAL STEPS/THOUGHTS INVOLVED IN THE GUS REPORTER BIOBRICK CLONING STRATEGY ~~

~~ THIS BIT REALLY NEEDS REMOVING AND RE-ORGANISING ~~

Morning

The group started thinking about their cloning strategies for each theme of the project. We also discussed idea for human practices and came up with the idea of an educational game based on synthetic biology. At the end of the day we had a group meeting to discuss our progress and our ideas for the modelling workshop for the UCL meet up.

BioGame

Today the game project was set up, created using the LibGDX-setup GUI in order to make projects for Android, Desktop and HTML deploym ent, all utilising LibGDX's built in features to run off a single "core" project. This was a big deciding factor in the choice to go with LIBGdx for developing the game as it allows us to quickly deploy across multiple platforms should we wish to do so. With the project set up, we have started to create assets such as textures and fonts to be loaded in and used within the game.

HBsu BioBrick Cloning Strategy

Plan to clone it into pSB1C3 then PCR the insert before integrating it into the amyE integration plasmid (pSB_Bs1C). Discovered that the Kanamycin resistance is an E.coli version and wouldn't work in the B. subtilis. Work is also being put into finding different antibiotic markers that would work in B.subtilis.

• Cloning strategies 2
• BioGame
• HBsu BioBrick Cloning Strategy

Morning

We continued work on the cloning strategies as well as starting modelling the subtilin two component system for the UCL workshop and the gus reporter system using BioNetGen. We also researched how L-Forms are made through research papers.

Afternoon

We produced a preliminary model of the gus reporter system. However, this model needs to be modified by adding parameters. We have also looked through some papers to find parameters required for the model.

BioGame

The Asset class has been created and all currently designed assets loaded into the game. A very simple (and temporary) cartoon bacteria has been loaded in and displayed on the screen along with the team logo. The pre-loading screen itself has not yet been developed but all constructs to create it are now in place. The percentage of loading done is simply outputted to the Java console for debugging purposes..

HBsu BioBrick Cloning Strategy

Designed Primers to be used in the Golden Braid assembly, and checked the melting temperature and self-annealing to make sure they work perfectly. Uses RFP (BBa_E1010) and GFP (BBa_E0040) to conjugate it to the HBsu protein in order to observe the recombination. Get a PCR protocol and Gel extraction protocol.

• Cloning strategies 3
• HBsu BioBrick Cloning Strategy

Morning

We further developed our cloning strategies and decided on the basic design of our construct. We made a preliminary design of the construct using Gene Designer to give a visual representation of how our construct should look like. From this image, we could see the positioning of all the parts, which will be more convenient for us when we want to devise a method to produce this.

We also improved the logo so it's ready for T-shirt printing.

HBsu BioBrick Cloning Strategy

A discussion with a supervisor revealed that the assembly system might be very time consuming. Ultimately, the decision was made to use DNA 2.0 to synthesis the HBsu BioBrick.

• Groningen plasmid Mini-prep
• BioGame
• HBsu BioBrick Cloning Strategy

Groningen plasmid Mini-prep

We performed a mini prep of the Groningen 2012 integration plasmid DNA grown in E.coli overnight at 37 degrees. The purity was tested using a NanoDrop. Introduction to microfluidics. Start work on designing Master Molds. Check the extracted pDNA to confirm the outcome of the purification of plasmid using the electrophoresis.

The plasmid has been cut with fast digest XbaI and BamHI to confirm the presence of the plasmid in the purified pDNA. Plasmid which has been cut by XbaI or BamHI only gives a linear piece of DNA. The gel shows that well with the DNA cut with both enzymes contains two pieces of DNA. One - plasmid backbone and the other - RFP reporter gene previously inserted into the plasmid. This confirms its identity.

BioGame

Further work on the game architecture has been done today. The main BioBrick class been created to provide common methods that all BioBricks will use. A very basic BioBrick has been created, and can be added upon touch to the Bacteria. This is simply to demonstrate what will happen when a BioBrick is added and the possibilities therein.

HBsu BioBrick Cloning Strategy

Due to the high cost of sequencing, the integration method of the HBsu BioBrick is now to clone the gene construct into the pMutin4 plasmid and use a single cross-over via the amyE gene to integrate it into the bacteria genome. Also the antibiotics marker and promoter is no longer being used because after SCO the whole plasmid will be integrated and as such will contain the promoter Pspac and Kanamycin cassette.

• Preparation for UCL
• B.subtilis Transformation
• Microfliuidics Introduction
• HBsu BioBrick Cloning Strategy

Preparation for UCL

We started preparing for our project presentation at UCL, and we have also started to prepare a presentation for our workshop about BioNetGen modelling.

B.subtilis Transformation

   ~~ DETAILS NEEDED HERE ~~

Microfluidics Introduction

Our iGEM team attended a talk about microfluidics given by Sunny Park. Microfluidics is vital to some of our sub-projects, therefore we looked into mechanism of operation and possible used including creation of designs for master moulds on the silicon wafer.

HBsu BioBrick Cloning Strategy

Sent the HBsu-GFP and HBsu-RFP BioBricks off for synthesis. Then, created an overnight liquid culture of LR2 rod B.subtilis cells into NB/MSM media to be used for creating L-form plates and getting microscopic pictures.

• Preparation for UCL
• Transformation
• Growing inducible L-form Bacillus subtilis
• Creating L-forms

Preparation for UCL

The team continued work on our modelling presentation for the UCL meetup. We added information about ordinary differential equation vs stochastic modelling and about Markov chain.

Transformation

The preparation of competent B.subtilis 168 was finished and the cells were transformed with the Groningen plasmid and streaked them out on the plates with negative controls. We also made some frozen competent B.subtilis 168 in glycerol.

Growing inducible L-form Bacillus subtilis

   ~~DETAILS NEEDED HERE~~

• Preparation for UCL
• Transformation Prep
• Growing inducible L-form B.subtilis

Preparation for UCL

   ~~ DETAILS NEEDED~~
   

Transformation Prep

Unfortunately the results of the Wednesday transformation have shown no growth on the chloramphenicol plasmid apart from a single colony.

Growing inducible L-form B.subtilis

   ~~ DETAILS NEEDED ~~

• Transform B.subtilis 168 with Groningen 2012 plasmid and RFP
• list item two

In attempts to characterise the Groningen 2012 integration plasmid we tried to transform B.subtilis 168 once again to check for integration at the SacA gene. The transformation was done using the protocol for competent Bacillus subtilis with positive control using the RFP plasmid and negative control with the water. The colonies were selected for using the chloramphenicol agar plates.

• Quick Check up on cultures and plates.

Put any details you want here. Paragraphs... Lists... Images... Go nuts :)

• Check on the cultures

There needs to be details here

• Grow B.subtilis 168 for transformation
• Set up the sucrose test
• Planting Seeds

Prep for Transformation

Inoculated 5 ml of freshly prepared MM media with B.subtilis 168 colonies. to grow overnight

Sucrose Test

To check whether the colony on the Cm plate with B.subtilis 168 containing Groningen 2012 integration plasmid actually contained transformant cells rather than a random mutant/contaminant we have decided to perform a sucrose metabolic test, by transferring the culture of into LB + 1% sucrose media and leaving it to grow over night.

Planting Seeds

• Transforming B.subtilis 168
• Results of sucrose test

Sucrose Test

We have checked the pH of the solution of cells from the Cm plate with Groningen 2012 integration plasmid incubated in sucrose rich media overnight to check for presence of acidic metabolites of sucrose and hence the activity of AmyE. The results were not comforting, the pH of the both samples we have prepared was the same as the pH of the +ve control (i.e. Bacillus 168 cells): 4.7, 4.8, 5.04.

Transformation

We have finished transforming B.subtilis 168 cells with Groningen 2012 integration plasmid and with pGMP (+ve control) and no plasmid at all (-ve control). Plated the colonies out according to the protocol and left to grow over night.

• Check the results of transformation
• Set up the test for Amylase

Transformation Results

Still no colonies on the chloramphenicol plates B.subtilis 168 with Groningen 2012 plasmid, however there were a few colonies on the positive control B.subtilis 168 with pGMP integration plasmid plates and no colonies on B.subtilis 168 LB + chloramphenicol plates.

Amylase Test

Just to make sure that the pGMP plasmid was integrated in all of the colonies, we have transferred a few colonies onto a freshly made LB + Starch agar plates for tomorrow, to check the amylase function using the iodine test for starch.

• Modelling Presentation
• Checking starch plates

Modelling Presentation

We made a few changes to our modelling presentation and practised our delivery in preparation for the presentation to members of the school of bioinformatics.

Checking Starch Plates

The starch plates were checked from yesterday and it was found that they were overgrown. So more starch agar plates were made including the antibiotic chloramphenicol (Cm) in the media. After these plates were made, colonies were transferred directly onto the plates and were left overnight.

• Workshop Practice Presentation
• Starch test

Workshop Practice Presentation

We gave a workshop practice presentation to members of the school of bioinformatics. It was decided that we shouldn't talk about the subtilin two-components system model, because it might be too complex for our workshop. It was suggested that our workshop needed to be a little less like a presentation, with more interaction for the participants.

Starch test

The starch plates were checked and a lawn of bacteria was found. The starch test was done anyway using vaporised iodine. The test showed a positive result for pGFP as it turned black showing no amylase was present and there for it had transformed. The B.subtilis (positive control) did not go black because the starch was consumed by the bacteria as expected. Also E.coli was left with the Groningen plasmid in 10ml of LB over the weekend for purification. This was done because stocks of the Groningen plasmid were running low.

• Updated SGM tutorial
• Finalized workshop presentation

SGM tutorial

We incorporated a section in the SGM tutorial about modelling. It is difficult deciding what information is best to include, and what to leave out. We were also thinking about using same exercises we are going to use in the workshop.

UCL Presentation Practice

We went through the UCL presentation with everyone on the team. After the presentation, everyone on the team gave feedback on how to improve certain slides and made note of what changes to make.

Workshop presentation

The slides for the workshop were edited accounting for Friday's feedback, and the finishing touches made. We also began producing a handout with exercises to complete during the workshop.

We incorporated our modelling into the SGM tutorial

• Making Plant Agar Plates
• Sucrose test set up

Making Agar Plates for our Plants

Today, we made up some Murashige and Skoog Medium (M&S Medium). This medium was ordered in powder form and water had to be added to it to be made into a solution. The solutions were then autoclaved to be used to make the agar for growing plants. After the solutions are done autoclaving, we made some agar with the M&S Medium. We weighed out 3.0g of agar no.2 into 2x500mL bottles. 250mL of M&S Medium was then added and mixed. The agar was then autoclaved and then cooled at 50 degree Celsius. When it is ready to be poured, we poured the agar onto 25 plates and left it to set.

Sucrose test set up

The plates from Thurs 11th were checked and inconclusive results were found as the agar was cloudy and individual colonies were not clear. For this reason a sucrose test was set up to see if the cells containing the Groningen plasmid from the plates (from Thurs) had integrated the plasmid, consequently interrupting the sacA gene and so failing to metabolise sucrose and showing as more basic. For a positive control we used B.subtilis and sucrose (this was expected to be acidic) and our negative control was LB and sucrose (this was expected to be more basic as no metabolism). These cultures were left overnight.

• Modelling Workshop
• Plasmid mini prep

Modelling Workshop Feedback Form

We produced a feedback form for attendees of our Workshop. This will be useful to access our workshops success and to improve its content for any future occasions we deliver a modelling workshop.

Plasmid mini prep

The Groningen plasmid was purified using plasmid mini prep of previously prepared E.coli. The purity was checked using the NanoDrop and a result of 183.7 was obtained meaning the DNA was usable in subsequent protocols. Also preparation for B.subtilis transformation was carried out; 5 ml of freshly prepared MM media was inoculated with B.subtilis 168 colonies to grow overnight.

• B.subtilis transformation

B.subtilis transformation

The B.subtilis transformation was carried out again to see if it would successfully integrate the Groningen plasmid when a lower concentration of Cm antibiotic is used in the plates. The plates used include:

• B.subtilis transformed with 10µl Groningen plasmid on 5µg/ml Cm, 12.5µg/ml Cm, Amp and LB
• B.subtilis transformed with 20µl Groningen plasmid on 5µg/ml Cm and 12.5µg/ml Cm
• B.subtilis transformed with 10µl pGFP (positive control) on 5µg/ml Cm, 12.5µg/ml Cm
• B.subtilis with water (negative control) on 5µg/ml Cm, 12.5µg/ml Cm and Amp

• Making sucrose and glucose agar

Making sucrose and glucose agar

The transformation plates were checked. Both sucrose and glucose agar was made, left to cool, plated and left to set. Then the colonies from the transformation plates were plated out onto the agar that was made. Both B.subtilis with the Groningen plasmid and B.subtilis alone (negative control) were put onto both sucrose and glucose plates.

• YSB 1.0 Conference

YSB 1.0 Conference

• Went down to London for a conference with all the other UK iGEM
• Gave a workshop on Rule Based Modelling
• Put up our poster- and got asked a lot of questions
• Listened to other teams presentations
• Spent the evening with the other teams, eating pizza and having a pub quiz

• Presentation

• We gave our presentation on L-forms! It went as expected and was well received.
• The other UK teams's presentations were also really good

• Sucrose test
• Transformation preparation
• Planting Seeds
• Shape-shifting modelling

Sucrose test

The sucrose test was carried out on the overnight cultures using a PH meter. The results observed were as follows:

• LB + sucrose + B.subtilis = 5.15
• LB + B.subtilis = 6.77
• LB + Groningen plasmid + sucrose = 4.23
• LB + Groningen plasmid = 6.75
• LB + sucrose = 7.07

These results show that cells with the Groningen plasmid had functional sucrase showing the plasmid has not been integrated so colonies must have been contaminants.

Transformation preparation

Preparation for B.subtilis transformation was carried out; 5 ml of freshly prepared MM media was inoculated with B.subtilis 168 colonies to grow overnight. Also E.coli with Groningen plasmid was put in LB media and left to grow overnight.

Planting Seeds

We washed and planted cabbage and french dwarf bean seeds in preparation for the introduction of L-forms into seedlings. The seeds were planted in Murishage and Skoog containing petri dishes and placed in a dark cuboard to germinate.

Shape-Shifting modelling

Modelling tasks for the shape-shifting team were identified:

1. Model cell shape using cytoskeleton structure
2. Model cell growth
3. Limit the growth of the cell by a certain shape (star or triangle)
4. OPTIONAL Change the membrane rigidity parameters and run the simulation again.

The shape of the cell is influenced by membrane fluidity and structure of the cytoskeleton. As cytoskeleton is present in both eukaryotic and prokaryotic cells. The former cells have no cell wall, so it is reasonable to assume that the shape of the L-form cell would obey similar rules as eukaryotic cell would. It has been determined that cytoskeleton behaves as an isotropic fluid. Therefore it's realsonable to rely on two-phase flow modelling frame (Holmes, Edelstein-Keshet, 2012).

• Innoculating Plants with L-forms
• Groningen Plasmid Mini-prep
• B.subtilis Transformation

Innoculating Plants with L-forms

We checked the seeds we planted yesterday and observed that the cabbage seeds had begun to germinate. This meant that they were ready to absorb L-forms. We soaked 5 cabbage seeds for 3 hours in 5ml of L-form solution and another 5 seeds in 5%(w/v) mannitol solution to act as a control. The seeds were then washed with distilled water (to lyse extracellular L-forms) and replated on Murishage and Skoog agar plates.

Groningen Plasmid Mini-prep

We performed a mini prep of the Groningen 2012 integration plasmid DNA grown in E.coli overnight at 37 degrees. This was done in 8 micro centrifuge tubes. The purity of each was tested using a NanoDrop.The average purity of the 8 tubes was 324ng/µl.

B.subtilis Transformation

The B.subtilis transformation was carried out again to see if it would successfully integrate the Groningen plasmid when a higher concentration of the plasmid was used. The plates used include:

• pGFP (positive control)on 5µg/ml Cm and 12.5µg/ml Cm

• Groningen plasmid 30 µl on 5µg/ml Cm and 12.5µg/ml Cm

• Groningen plasmid 45 µl on 5µg/ml Cm and 12.5µg/ml Cm

• Groningen plasmid 60 µl on 5µg/ml Cm and 12.5µg/ml Cm

• Water (negative control) on 5µg/ml Cm and 12.5µg/ml Cm

• Check the Transformation Plates
• Modelling

Transformation results (Groningen plasmid)

The results of the last transformation of B.subtilis with Groningen 2012 integration plasmid look promising. Everything has worked as expected. The insignificant difference in the number of colonies grown on plate with cells inoculated with different amount of plasmid indicates that the transformation occurred as efficiently as it could at around 9ug of DNA per reaction tube. The reason why nothing grew on the plates with 12.5ug/ml of chloramphenicol may be the inefficiency of the promoter used. The colony counts are recorded in a table below.

Transformation results

Agar/Transformants	pGFP (positive control)	Groningen plasmid 30 µl	Groningen plasmid 45 µl	Groningen plasmid 60 µl	Water
Nutrient Agar + 5µg/ml Cm	134 Colonies	5 Colonies	9 Colonies	7 Colonies	0 Colonies
LB Agar = 12.5µg/ml Cm	132 Colonies	0 Colonies	0 Colonies	0 Colonies	0 Colonies

To confirm the integration of the plasmid the test for sucrose metabolism will be carried out on Monday.

Photos to follow.

Modelling

Preparation work for modelling, learning to use VCell software. We started working our way through simple tutorials to get more familiar with the program before doing "real work".

The structure and function of cytoskeleton was studied. The cell behaves like an isotropic fluid in terms of shape and movement, therefore some of the factors which influence the shape could be identified: Cell Volume, surface area, surface tension, hydrostatic pressure.

It turns out that eukaryotic cytoskeleton is characterised as a two-phase viscous, responsive and contractile liquid. If bacterial cytoskeleton is similar, it would complicate the modelling by a fair bit. More insight into the structure and physical properties of bacterial cytoskeleton is needed. In terms of cell growth and division it will be useful to look into the properties of FtsZ and MreB as well as properties of the vesicular membrane and physical triggers of the process of 'blebbing'. So that's a job for Monday.