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Team:Warsaw/Project
Escherichia coli, which is by far the greatest model for iGEM projects, is a gram-negative bacterium. Because of that, the expression of some proteins, which came from gram-positive bacteria, is sometimes hard to achieve in E. coli. Even if we managed to express proteins from gram-positive bacteria in a gram-negative model, the proteins could for example have different localization in the cell and behave differently from how they did in the original cell they came from.
Bacillus subtilis is a great model of gram-positive bacteria; it was also used by some iGEM teams in previous years. We were curious, why only a few teams used that model. When we began searching partsregistry for some BioBricks especially for Bacillus subtilis, we realized that there is only a very limited number of them. It could be the reason why this model isn't so popular, but it is also the reason why we found working with Bacillus subtilis so interesting and at the same time so daring and challenging.
Of course, we wanted to make a functional project, but we also thought that it is a great challenge to work with another model than E. coli. We truly believed, that making some new BioBricks for Bacillus subtilis, even such basic ones like promoters and rbs, is important because it would help the iGEM community to work with this great model of gram-positive bacteria in the future.
Our goal this year was to create a bacteria strain which could carry certain genes into eucaryotic cells. For safety reasons, and also to keep the work more coherent, we divided our task into two separate steps:
The first one was creating an 'invasive' Bacillus subtilis strain. Bacillus is a non-pathogenic bacteria living peacefully in the soil. However, there are many bacteria species that have the ability to invade animal as well as human cells. We had an idea to create a plasmid for B. subtilis carrying listerolysin gene as to enable the bacteria to enter the eucaryotic cells just as Listeria monocytogenes from which the gene was taken does. L. monocytogenes is a dangerous pathogen; however, B. subtilis is a safe bacterium. With the lysis device installed in it, B. subtilis cells lyse after entering the eucaryotic cells. Since both of the bacteria are gram-positive, gene expression should undergo without obstructions. The plasmid also carries the GFP coding device which will help us to determine the success of our experiment. After the lysis of B. subtilis cells, GFP will be released and the measurement of its fluorescence will give us the idea of how the experiment worked out.
The second step was creating a shuttle vector which would be capable of replication and gene expression inside the eucaryotic cells. The vector will carry RFP coding device, and it will be RFP flourescence measurement which will confirm the success of our experiment. However, due to safety reasons, we did not combine the two steps of our experiment. Each of the systems is tested separately, with all the safety precautions. In the future, when all the safety issues are resolved, these systems might be able to help a great deal in gene therapy. Delivering certain genes right into the malfunctioning cells would be a tremendous help in treatment of complicated diseases.
In the first step of our project we designed few BioBricks adapted for Bacillus subtilis. We decided, that even if some basic parts are available in partsregistry, we would create a few more to expand the amount of BioBricks for this bacterium. Therefore, we designed:
- Two new promoters for Bacillus subtilis
- Three new rbs for Bacillus subtilis
- One terminator for Bacillus subtilis
We believed that these parts will be useful for creating constructs designed for Bacillus subtilis in our this year's project and in the future projects based on this bacteria.
We performed all designing in Clone Manager program, which we found easy and useful.
Then we designed parts especially devoted to our project. Our goal was to create two main constructs:
First construct should allow Bacillus subtilis to enter mammalian cell. To give Bacillus this ability, we decided to use listeriolisin, protein naturally existing in Listeria monocytogenes. To confirm, that Bacillus entered the mammalian cell we used GFP protein, which would give us a fluorescent signal, easy to notice by using fluorescent microscopy.
We designed our construct like this:
promoter + rbs + GFP + terminator + promoter + rbs + Listerolysin + terminator
Especially for this construct we designed Listerolysin part devoted to usage in Bacillus subtilis.
Our plan was to test all our basic parts in this construct, by creating few parallel versions, using different promoters and rbs and choose the best version.
Our second goal was to create a plasmid which could replicate and express in both, bacterial and mammalian cell. We based our plasmid on pSB1a3 plasmid backbone.
To allow plasmid to replicate in a mammalian cell we designed special BioBrick containing oriP and EBNA1 gene from Epstein barr virus. To confirm expression of the plasmid in mammalian cell we planed to use RFP protein under control of a mammalian promoter CMV. To confirm expression in E. coli cells we used construct witch GFP.
Second main construct was devoted to use directly in mammalian cell. It was based on BioBricks from partregistry witch our new part containing oriP.
super folder GFP + CMV promoter + rbs (Kozak sequences) + RFP + oriP
We designed the oriP part especially for this construct.
Second step
Then we entered the wet lab and started preparing our BioBrick made of DNA, not only on the computer screen ;)
We decided to achieve our promoters, rbs and terminator parts with method called in our laboratory “PCR without template”. It goes like this:
- For each part we designed two starters that covered each other in about 20bp on 3' ends
- Each starter had prefix or suffix (depending on the starter) on it's 5' end
- Then we prepared PCR reaction mixture
- We used Taq polymerase mastermix (nazwa???) and primers in concentration
- We didn't add any DNA template
- We calculated the annealing temperature by oligocalculator
- For first three cycles of PCR reaction temperature was calculated for primer without overhang
- Then for thirty more cycles temperature of annealing was calculated for the whole long primer with overhang – that is because from this moment there was enough product of the reaction in the reaction mix to become a template for the next cycles
- And that's it!
Then we cloned our PCR product into pJet plasmid, using special kit for cloning PCR products (linka?), and transformed with it E. coli TOP10. After isolation of the plasmid, we confirmed the sequence of received product by DNA sequencing (traditional Sanger sequencing). From this step, our parts were ready to digest and clone with procedures recommended by iGEM :)
We managed to optimize reaction conditions and received all of ours parts by PCR reaction. Unfortunately, then we faced some problems and weren't as successful as we hoped. We didn't manage to confirm all our products' sequences by DNA sequencing. Finally we received a full BioBrick part:
- Promoter
- Two RBS
- Terminator
Third step
As soon as we received first full and confirmed BioBrick part, we started preparing our construct for Bacillus subtilis. We began from ligation of the part with GFP, because that would allow us to check if our promoter and RBS works. We used 3A assembly procedure, witch we found on iGEM website. We followed the set pattern:
Step four
When we had a ready construct consisting of promoter + RBS + GFP we cloned it on pTG262 vector. This plasmid, which was constructed by Edinburgh iGEM team in 2007, is the only plasmid meeting iGEM standards designed especially far Bacillus subtilis. We managed to successfully clone our insert to the vector, what we confirmed by digesting with EcoRI and PstI and gel electrophoresis of the vector and insert.
21. If any one break a hole into a house (break in to steal), he shall be put to death before that hole and be buried.
22. If any one is committing a robbery and is caught, then he shall be put to death.
23. If the robber is not caught, then shall he who was robbed claim under oath the amount of his loss; then shall the community, and ... on whose ground and territory and in whose domain it was compensate him for the goods stolen.
24. If persons are stolen, then shall the community and ... pay one mina of silver to their relatives.
25. If fire break out in a house, and some one who comes to put it out cast his eye upon the property of the owner of the house, and take the property of the master of the house, he shall be thrown into that self-same fire.
26. If a chieftain or a man (common soldier), who has been ordered to go upon the king's highway for war does not go, but hires a mercenary, if he withholds the compensation, then shall this officer or man be put to death, and he who represented him shall take possession of his house.
27. If a chieftain or man be caught in the misfortune of the king (captured in battle), and if his fields and garden be given to another and he take possession, if he return and reaches his place, his field and garden shall be returned to him, he shall take it over again.
28. If a chieftain or a man be caught in the misfortune of a king, if his son is able to enter into possession, then the field and garden shall be given to him, he shall take over the fee of his father.
29. If his son is still young, and can not take possession, a third of the field and garden shall be given to his mother, and she shall bring him up.
30. If a chieftain or a man leave his house, garden, and field and hires it out, and some one else takes possession of his house, garden, and field and uses it for three years: if the first owner return and claims his house, garden, and field, it shall not be given to him, but he who has taken possession of it and used it shall continue to use it.
