"
Team:Dundee/Notebook
From 2012.igem.org
Notebook
Week One
Biology
The first week for the biologists was a general introduction to the ways of the lab. We were given a health and safety briefing and attended a health and safety seminar (an experience that would terrify any undergrad new in the lab). The team was taken through all the experiments that would be crucial for the project and a general plan of attack was devised. The main achievements of this week were the production of competent DH5α E. coli cells; purification and amplification of Salmonella typhimurium LT2 chromosomal DNA and digestion of pUNI-PROM with EcoRI and BamHI or XbaI BamHI.
Maths
The Mathematics team began by looking at common modelling methods in biology. After some research, we started to piece together a system of ordinary differential equations (ODEs) representing the populations of E. coli and C. difficile using logistic growth models.
Software
The team created a template Blog website and Twitter account. To provide a graphical representation of the project, existing software modelling options were investigated to ascertain whether an existing tool could provide suitable functionality or if a bespoke solution would be preferable. Work commenced on an Android framework for a suite of mobile utilities that will be developed over the course of the project.
The first week for the biologists was a general introduction to the ways of the lab. We were given a health and safety briefing and attended a health and safety seminar (an experience that would terrify any undergrad new in the lab). The team was taken through all the experiments that would be crucial for the project and a general plan of attack was devised. The main achievements of this week were the production of competent DH5α E. coli cells; purification and amplification of Salmonella typhimurium LT2 chromosomal DNA and digestion of pUNI-PROM with EcoRI and BamHI or XbaI BamHI.
Maths
The Mathematics team began by looking at common modelling methods in biology. After some research, we started to piece together a system of ordinary differential equations (ODEs) representing the populations of E. coli and C. difficile using logistic growth models.
Software
The team created a template Blog website and Twitter account. To provide a graphical representation of the project, existing software modelling options were investigated to ascertain whether an existing tool could provide suitable functionality or if a bespoke solution would be preferable. Work commenced on an Android framework for a suite of mobile utilities that will be developed over the course of the project.
Week Two
Biology
The second week was when the project really began. The first task was to amplify the 13 type VI secretion system genes by PCR. TssA, TssE, TssF, TssM, Hcp and VgrG were all cloned individually from the prepared S. typhimurium chromosomal DNA. ClpV, VipA, VipB and TssJ, TssK (referred to hereafter at ClpV and TssJK respectively) were cloned as the original operons. Following PCR, results were analysed on a gel and showed that every gene was successfully amplified. The genes were then PCR purified and digested either BglII- HindIII or BclI- HindIII (TssM). Hcp (needle) and VgrG (needle tip) genes were cloned as fuse genes, i.e. they were cloned without a stop codon to allow the addition of another gene. These fuse genes were digested BglII- EcoRI. Digests were strata-cleaned, ligated into pUNI-PROM and transformed into DH5α cells. Transformed colonies were mini-prepped, digested EcoRI-HindIII to remove the insert and products were run on a gel. Clones TssA, TssE, TssJK, Hcp and Hcp Fuse appeared correct and were sent for sequencing. TssF, VgrG, VgrG fuse and ClpV failed to show an insert on the gel so were rechecked using colony PCR.
Maths
We continued to work on our interacting species model by including specific parameters, such as removal rates due to the natural processes in the colon, competition terms and the death rate of C. difficile due to the engineered type VI secretion system in E. coli.
Software
Testing of a promising software modelling tool proved disappointing due to its inability to represent large cell number interactions. After consideration, the decision was made to begin development of a bespoke C++ application that could handle realistically large colonies of cells, whilst integrating the mathematical modelling aspects into the behaviour. The aim is to provide a cross-platform, cellular automata model of the project features top provide a graphical means to explore the project’s biological dynamics. Work also began on developing the team’s official Wikipedia website.
The second week was when the project really began. The first task was to amplify the 13 type VI secretion system genes by PCR. TssA, TssE, TssF, TssM, Hcp and VgrG were all cloned individually from the prepared S. typhimurium chromosomal DNA. ClpV, VipA, VipB and TssJ, TssK (referred to hereafter at ClpV and TssJK respectively) were cloned as the original operons. Following PCR, results were analysed on a gel and showed that every gene was successfully amplified. The genes were then PCR purified and digested either BglII- HindIII or BclI- HindIII (TssM). Hcp (needle) and VgrG (needle tip) genes were cloned as fuse genes, i.e. they were cloned without a stop codon to allow the addition of another gene. These fuse genes were digested BglII- EcoRI. Digests were strata-cleaned, ligated into pUNI-PROM and transformed into DH5α cells. Transformed colonies were mini-prepped, digested EcoRI-HindIII to remove the insert and products were run on a gel. Clones TssA, TssE, TssJK, Hcp and Hcp Fuse appeared correct and were sent for sequencing. TssF, VgrG, VgrG fuse and ClpV failed to show an insert on the gel so were rechecked using colony PCR.
Maths
We continued to work on our interacting species model by including specific parameters, such as removal rates due to the natural processes in the colon, competition terms and the death rate of C. difficile due to the engineered type VI secretion system in E. coli.
Software
Testing of a promising software modelling tool proved disappointing due to its inability to represent large cell number interactions. After consideration, the decision was made to begin development of a bespoke C++ application that could handle realistically large colonies of cells, whilst integrating the mathematical modelling aspects into the behaviour. The aim is to provide a cross-platform, cellular automata model of the project features top provide a graphical means to explore the project’s biological dynamics. Work also began on developing the team’s official Wikipedia website.
Week Three
Biology
From this week onwards, the Biology tasks were split between the four members of the lab team.
Combinatorial clone
TssA was the first gene to be fully sequenced and as a result was chosen as the first gene in the combinatorial clone. The plasmid containing TssA was digested BamHI-HindIII so that the next gene could be added by suicide ligation at the Bam site. Following digestion, the plasmid was ran on a gel and then extracted. Ligations were performed adding TssJK and ClpV and transformations were completed using DH5α cells. Plates were left overnight and then liquid cultures were set up. These were digested EcoRI- HindIII to remove the insert and ran on a gel. TssA+ClpV was sent for sequencing using a primer designed from the latter part of TssA. Once this had been checked, TssA+ClpV was digested BamHI- HindIII and TssJK was added.
Site directed mutagenesis
The type VI secretion system genes TssL and TssG required SDM. This was due to the presence of an EcoRI site in the TssL gene and both EcoRI and PstI sites within the TssG sequence. These genes were amplified and digested HindIII only. Plasmid pSB1C3 was prepared and also digested HindIII. TssL and TssG were ligated into pSB1C3 and transformed into DH5α cells. However, no inserts were found in the initial minipreps prepared and so patch plates were made for both genes.
Fuse genes
mCherry and endolysin genes were PCR’d and digested XbaI- HindIII ready for ligation to Hcp Fuse and VgrG Fuse genes.
Maths
We analysed our model using MATLAB to create graphs illustrating how the populations change over time. An online phase plane application was used in order to create phase plane diagrams showing the steady states of the ODE system. We first considered the model excluding the removal rates to gain an idea of how the species may interact in laboratory conditions. Once satisfied with our initial results, we then included the removal rates to encompass the real world biological behaviour.
Software
Development of the cellular automata tool continued, and now provides generation of randomly placed cells as well as pseudo-Brownian motion cell behaviour. Current testing proved to handle 250’000 interacting cells. A text-book implementation of the MVC (model-view-controller) architecture has been employed, though bottlenecks have resulted. Code has been profiled using Valgrind & Callgrind to identify areas for code refactoring.
From this week onwards, the Biology tasks were split between the four members of the lab team.
Combinatorial clone
TssA was the first gene to be fully sequenced and as a result was chosen as the first gene in the combinatorial clone. The plasmid containing TssA was digested BamHI-HindIII so that the next gene could be added by suicide ligation at the Bam site. Following digestion, the plasmid was ran on a gel and then extracted. Ligations were performed adding TssJK and ClpV and transformations were completed using DH5α cells. Plates were left overnight and then liquid cultures were set up. These were digested EcoRI- HindIII to remove the insert and ran on a gel. TssA+ClpV was sent for sequencing using a primer designed from the latter part of TssA. Once this had been checked, TssA+ClpV was digested BamHI- HindIII and TssJK was added.
Site directed mutagenesis
The type VI secretion system genes TssL and TssG required SDM. This was due to the presence of an EcoRI site in the TssL gene and both EcoRI and PstI sites within the TssG sequence. These genes were amplified and digested HindIII only. Plasmid pSB1C3 was prepared and also digested HindIII. TssL and TssG were ligated into pSB1C3 and transformed into DH5α cells. However, no inserts were found in the initial minipreps prepared and so patch plates were made for both genes.
Fuse genes
mCherry and endolysin genes were PCR’d and digested XbaI- HindIII ready for ligation to Hcp Fuse and VgrG Fuse genes.
Maths
We analysed our model using MATLAB to create graphs illustrating how the populations change over time. An online phase plane application was used in order to create phase plane diagrams showing the steady states of the ODE system. We first considered the model excluding the removal rates to gain an idea of how the species may interact in laboratory conditions. Once satisfied with our initial results, we then included the removal rates to encompass the real world biological behaviour.
Software
Development of the cellular automata tool continued, and now provides generation of randomly placed cells as well as pseudo-Brownian motion cell behaviour. Current testing proved to handle 250’000 interacting cells. A text-book implementation of the MVC (model-view-controller) architecture has been employed, though bottlenecks have resulted. Code has been profiled using Valgrind & Callgrind to identify areas for code refactoring.
Week Four
Biology
Combinatorial clone
TssA+ClpV+TssJK was digested Eco Hind and sent for sequencing. The plasmid was then digested (BamHI- HindIII) and TssE was added by ligation and transformation. When this was successful, the procedure was repeated and TssA+ClpV+TssJK+TssE was sent for sequencing.
Site directed mutagenesis
TssL patch PCR was successful as an insert was found so this was prepared and sent for sequencing. TssG still had not worked so the PCR was repeated and the product digested as before. Also screened some more patches in case an insert had been missed but these were unsuccessful.
Fuse genes
Western blots were carried out using VgrG, VgrG fuse and Hcp fuse cell and supernatant samples. mCherry and endolysin PCR was repeated and ligations to VgrG and Hcp Fuse were carried out and transformed with JM1061 cells. Hcp fused with endolysin was successful.
Maths
To create a more complex and realistic model, we decided to use partial differential equations (PDEs). PDEs can be used to illustrate not only how the population of the bacteria change in time but also in space, which enabled us to model real life situations, where the C. diff would be adhered to the walls of the colon. We began by analysing these models on MATLAB and COMSOL using basic geometry as well as including diffusion terms.
Software
Analysis of the cellular automata program has identified a bottleneck that results from cell iteration being executed by both the model and view. The code was therefore refactored to exploit the graphics surface as a partial data structure, which relieves the bottleneck by updating the model and rendering new cell positions in a single iteration. This refactoring has increased cell handling capability to 300’000. Code to emulate physics behaviour has begun, and flow modelling has been integrated into the code.
Combinatorial clone
TssA+ClpV+TssJK was digested Eco Hind and sent for sequencing. The plasmid was then digested (BamHI- HindIII) and TssE was added by ligation and transformation. When this was successful, the procedure was repeated and TssA+ClpV+TssJK+TssE was sent for sequencing.
Site directed mutagenesis
TssL patch PCR was successful as an insert was found so this was prepared and sent for sequencing. TssG still had not worked so the PCR was repeated and the product digested as before. Also screened some more patches in case an insert had been missed but these were unsuccessful.
Fuse genes
Western blots were carried out using VgrG, VgrG fuse and Hcp fuse cell and supernatant samples. mCherry and endolysin PCR was repeated and ligations to VgrG and Hcp Fuse were carried out and transformed with JM1061 cells. Hcp fused with endolysin was successful.
Maths
To create a more complex and realistic model, we decided to use partial differential equations (PDEs). PDEs can be used to illustrate not only how the population of the bacteria change in time but also in space, which enabled us to model real life situations, where the C. diff would be adhered to the walls of the colon. We began by analysing these models on MATLAB and COMSOL using basic geometry as well as including diffusion terms.
Software
Analysis of the cellular automata program has identified a bottleneck that results from cell iteration being executed by both the model and view. The code was therefore refactored to exploit the graphics surface as a partial data structure, which relieves the bottleneck by updating the model and rendering new cell positions in a single iteration. This refactoring has increased cell handling capability to 300’000. Code to emulate physics behaviour has begun, and flow modelling has been integrated into the code.
Week Five
Biology
Combinatorial clone
TssA+ClpV+TssJK+TssE was digested and ligated with TssF. Transformation was successful and the plasmid was sent for sequencing. Sequencing results showed a problem with TssF so this ligation was repeated. Following 2 further unsuccessful attempts a successful transformation was finally produced.
Site directed mutagenesis
TssL sequencing came back showing that it was correct and so SDM was begun on this gene, following the quickchange procedure. Retried PCR on TssG but this failed twice and so went back to check the previous digests of this gene and set up new ligations, which were transformed into DH5α cells. TssL SDM product was sent for sequencing, as after digesting some of the minipreps and running on a gel, it looked as though the procedure had been successful.
Fuse genes
More western blots were carried out with the VgrG and Hcp genes in S. typhimurium and Hcp-endolysin in E. coli. VgrG fused with endolysin failed to work and so more ligations were set up.
Maths
Continuing to use COMSOL, we modified the geometry of the model by adding different domains. We allocated certain initial conditions to specific domains so that the model would coincide with the positions of the bacteria in the colon.
Software
The Android application was upgraded to provide a dilutions calculator which can run simple or serial dilution calculations. The cellular automata program was extended to encompass further physics attributes - nutrient levels (consumption and supply), cellular mitosis and tissue and mucus effects.
Combinatorial clone
TssA+ClpV+TssJK+TssE was digested and ligated with TssF. Transformation was successful and the plasmid was sent for sequencing. Sequencing results showed a problem with TssF so this ligation was repeated. Following 2 further unsuccessful attempts a successful transformation was finally produced.
Site directed mutagenesis
TssL sequencing came back showing that it was correct and so SDM was begun on this gene, following the quickchange procedure. Retried PCR on TssG but this failed twice and so went back to check the previous digests of this gene and set up new ligations, which were transformed into DH5α cells. TssL SDM product was sent for sequencing, as after digesting some of the minipreps and running on a gel, it looked as though the procedure had been successful.
Fuse genes
More western blots were carried out with the VgrG and Hcp genes in S. typhimurium and Hcp-endolysin in E. coli. VgrG fused with endolysin failed to work and so more ligations were set up.
Maths
Continuing to use COMSOL, we modified the geometry of the model by adding different domains. We allocated certain initial conditions to specific domains so that the model would coincide with the positions of the bacteria in the colon.
Software
The Android application was upgraded to provide a dilutions calculator which can run simple or serial dilution calculations. The cellular automata program was extended to encompass further physics attributes - nutrient levels (consumption and supply), cellular mitosis and tissue and mucus effects.
Week Six
Biology
Combinatorial clone
The plasmid containing TssA+ClpV+TssJK+TssE+TssF was mini-prepped and sent for sequencing. It was then digested and ligated with TssM. TssM failed to work and so the original PCR and digestion of this gene was repeated, as it had shown quite low yield from the start.
Site directed mutagenesis
TssL sequencing confirmed that the SDM had been successful and so this was now ready to be cloned in with the other genes in the combinatorial clone. TssG sequencing still showed the gene to be wrong and so gel purified the digest that had been used for ligations last week again and then set up new ligations which were transformed into DH5α cells. This did not work and so decided to try a different digest of the gene (Bgl II and Hind III) and clone it into a different vector (rather than the pSB1C3). Ligations were therefore set up using PUT18 and T7KS vectors. Transformations did not work correctly though and so set up some patch plates, to screen colonies using PCR.
Fuse genes
VgrG-endolysin, VgrG-mCherry and Hcp-mCherry all successfully ligated and sent for sequencing.
Maths
We altered the previously mentioned models, by using functions to set the initial conditions instead of specific domains. We also started to model the interaction between C. diff and endolysin, in order to determine the amount of endolysin required to kill one C. diff bacterium.
Software
The Android utility, now named ‘Lazy Scientist II’ was analysed for compatibility issues, and the code was refactored to allow the code to execute safely on a wider range of devices. Work began on an Android based game, with a preliminary MVC framework established ready for game logic to be added. Current application has placeholders for game logic and model aspects, and has a touch-screen handling routine that provides co-ordinates of screen presses.
Combinatorial clone
The plasmid containing TssA+ClpV+TssJK+TssE+TssF was mini-prepped and sent for sequencing. It was then digested and ligated with TssM. TssM failed to work and so the original PCR and digestion of this gene was repeated, as it had shown quite low yield from the start.
Site directed mutagenesis
TssL sequencing confirmed that the SDM had been successful and so this was now ready to be cloned in with the other genes in the combinatorial clone. TssG sequencing still showed the gene to be wrong and so gel purified the digest that had been used for ligations last week again and then set up new ligations which were transformed into DH5α cells. This did not work and so decided to try a different digest of the gene (Bgl II and Hind III) and clone it into a different vector (rather than the pSB1C3). Ligations were therefore set up using PUT18 and T7KS vectors. Transformations did not work correctly though and so set up some patch plates, to screen colonies using PCR.
Fuse genes
VgrG-endolysin, VgrG-mCherry and Hcp-mCherry all successfully ligated and sent for sequencing.
Maths
We altered the previously mentioned models, by using functions to set the initial conditions instead of specific domains. We also started to model the interaction between C. diff and endolysin, in order to determine the amount of endolysin required to kill one C. diff bacterium.
Software
The Android utility, now named ‘Lazy Scientist II’ was analysed for compatibility issues, and the code was refactored to allow the code to execute safely on a wider range of devices. Work began on an Android based game, with a preliminary MVC framework established ready for game logic to be added. Current application has placeholders for game logic and model aspects, and has a touch-screen handling routine that provides co-ordinates of screen presses.
Week Seven
Week7...
Week Eight
Week8...
Week nine
Week9...
Week Ten
Week10...
