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The gel electrophoresis of Jen1 (<partinfo>BBa_K284002</partinfo>), Dld (<partinfo>BBa_K284003</partinfo>) and VHb (<partinfo>BBa_K258005</partinfo>) was analyzed. Only two bands per column were expected, insert and backbone, but there was at least one extra at each column. The length of the genes Jen1, Dld and VHb are 998 bp, 463 bp and 137 bp, respectively, so the bands closest to the given sizes were cut out of the gel and purified using a QIAquick PCR Purification kit. The concentrations of the purified genes are listed below:
Gel electrophoresis was also performed on the lysis device and pBAD promoter. The expected sizes of the DNA sequences were 2079 bp for the lysis plasmid, 1785 bp for the lysis device and about 2000 bp for the pBAD promoter with backbone. This corresponds well with the observed bands. Pictures of the gels obtained from the two electrophoresis runs are included below. On both gels, the far left and far right wells contain GeneRuler 1kb DNA ladder.
The DNA segment containing the VGB promoter was extracted from the gel according to protocols.
The BioBricks colicin (<partinfo>BBa_K150009</partinfo>), dld-promoter (<partinfo>BBa_K284003</partinfo>), jen1 (<partinfo>BBa_K284002</partinfo>) and VHb-promoter (<partinfo>BBa_K258005</partinfo>) were miniprepped as described in the protocol. The concentration results are listed below:
The luxI BioBrick <partinfo>BBa_C0061</partinfo> was transformed. Continued work on circuit assembly using J5.
Restriction digestion was performed on dld-promoter (<partinfo>BBa_K284003</partinfo>), jen1-promoter (<partinfo>BBa_K284002</partinfo>) and VHb-promoter (<partinfo>BBa_K258005</partinfo>) as described in the protocol. All of them cut with E and S.
Restriction digest were also performed for the vgb-promoter (E + S) and RBS (E + X), and gel electrophoresis was performed to verify that the correct DNA fragment were made during digestion. The RBS digestion sample was purified using a QIAquick PCR Purification kit. Using this kit will cause small DNA fragments, like the prefix fragment between the EcoRI and XbaI sites, to be lost. All of the vgb sample was run on gel, to separate the insert to be further used, from the backbone.
Performed restriction digest on the pBAD (<partinfo>BBa_K206000</partinfo>) and lysis <partinfo>BBa_K112808</partinfo> parts, cutting pBAD with SpeI and PstI, and the lysis part with XbaI and PstI.
Wiki design updated. [http://j5.jbei.org/ J5] was used to determine how to assemble the genetic circuit. The total size of the plasmid (depending on the size of the backbone) will probably be 8000-9000 bp, of which the BioBricks make up about 6500 bp.
A warning appeared in the output when running the assembly in J5. It might be problematic to incorporate the LuxR-HSL promoter (<partinfo>BBa_R0062</partinfo>) in the circuit.
Also, it was discovered that the colicin BioBrick (<partinfo>BBa_K150009</partinfo>) consists of a number of DNA segments, not only genes for the necessary colE1 proteins. Among these are the luxR gene and the LuxR-HSL promoter as well as a lysis-inducing component. This means that inserting the entire BioBrick into our circuit will be problematic, as the LuxR production and LuxR-HSL sensitivity of the construct will interfere with the oxygen promoter and the rest of the luxR components in our circuit. The problem can probably be circumvented if we amplify the genes coding for the two colE1 proteins and connect them to a constitutive promoter.
Restriction digestion was performed on isolated DNA from the VGB and RBS biobricks (<partinfo>BBa_k561001</partinfo> and <partinfo>BBa_B0034</partinfo>) according to protocols. Gel electrophoresis was used to determine the size of the resulting fragments. The RBS restriction digest was filtered using the QIAquick PCR purificat kit. The results for VGB were not as expected, and restriction digest of this part was therefore performed again, this time with addition of [http://en.wikipedia.org/wiki/Calf-intestinal_alkaline_phosphatase CIP] to the digest solution at the end of the procedure.
Miniprepped all three tubes with pBAD inoculated yesterday. Tube 3 contained 10 mL culture and was divided into two tubes, so that all processed tubes contained ~5 mL culture. Results of DNA measurements were as follows (ng/uL):
Discarded B3-1 and B3-2, placed B1 and B2 in -20 C freezer.
In order to make a new plate of Lysis 1, transferred 20 uL from one of the tubes with liquid culture inoculated yesterday to one LA + Amp plate, and 50 uL to another. Placed the plates in the 37 C incubator cabinet. Also transferred 50 uL as inoculate to the tube used as negative control yesterday and placed it in the shaking incubator. Then combined all three tubes into one and placed in refrigerator to limit cell death before miniprep tomorrow.
Removed pBAD plates from 37 C incubator. Good growth on all transformed plates, Negative control showed no growth. Inoculated three colonies from plate B in liquid LB + Amp. Discarded the "mixed" plate. Placed plates A and B in refrigerator. Also inoculated three colonies from the lysis biobrick <partinfo>BBa_K112808</partinfo> using the plate from 22/6. Used an inoculating needle for all inoculations today.
LuxI (<partinfo>BBa_K092400</partinfo>) was transferred to petri dishes with Chloramphenicol and Kanamycin, to test if LuxI has a different resistance than given. Pbad (<partinfo>Bba_K206000</partinfo>) was transformed and transferred to petri dishes with Ampicilin. A new batch of LA-medium with Ampicilin was prepared and poured on to petri dishes. Extracted and transformed the pBAD strong promoter biobrick <partinfo>BBa_K206000</partinfo>.
Researched the ArcA protein and its binding sequence(s) to investigate whether the lldPRD operon promoter can be modified to eliminate repression by ArcA-P in the anaerobic state. Located a putative ArcA-P binding site in the promoter sequence: The sequence GTTAACTAAATGTTA is the reverse complement of the minus strand of the 85 bp promoter sequence from position 38 to 52. This sequence is identified by [http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1999.01347.x/full#t1 McGuire et al (1999)] (see [http://arep.med.harvard.edu/ecoli_matrices/dat/arcA.dat this] list). In the GenBank entry this part of the sequence reads TAACATTTAGTTAAC
Favorov et al. have made a newer suggestion for a general motif for ArcA binding sites - see [http://bioinformatics.oxfordjournals.org/content/21/10/2240.full#sec-5 this article] and the computational result [http://favorov.bioinfolab.net/SeSiMCMC/examples/arcA/result.html here]. According to Favorov, the crucial features of the site is a direct repeat, as shown below:
atacaTAACatttagtTAACcattc
Extracted the pBAD strong promoter biobrick <partinfo>BBa_K206000</partinfo> and transformed it in two samples. Plated out on three Amp plates as follows: 200 uL from sample A, 200 uL from sample B, 20 uL each from sample A and B.
A colony of LuxR (<partinfo>BBa_R0062</partinfo>) was transferred to liquid medium, but there were no colonies of LuxI (<partinfo>BBa_K092400</partinfo>). A new agar plate with LuxI was prepped.
Performed isolation of plasmid DNA from RBS (<partinfo>BBa_B0034</partinfo>), C78, C79 and VGF promoter (<partinfo>BBa_K561001</partinfo>). The parts coding for the LuxR and LuxI genes, <partinfo>BBa_R0062</partinfo> and <partinfo>BBa_K092400</partinfo> respectively, were transformed again.
Colonies from <partinfo>BBa_C0078</partinfo>, <partinfo>BBa_C0079</partinfo> and <partinfo>BBa_K561001</partinfo> were transferred from agar plates to liquid medium.
Performed isolation of plasmid DNA from the ribosome binding site (RBS) part <partinfo>BBa_B0034</partinfo>, and the double transcriptional terminator (DTT) part <partinfo>BBa_B0015</partinfo> grown on ampicillin and on kanamycin. The yield was quite poor, especially for RBS. We then decided to run the spin columns one more time to see if we could get a higher yield the second time. Since we are going to use a lot of RBS, we made a liquid culture right away, so we can perform a new DNA isolation on RBS tomorrow.
Wiki design and contents was revised. Front page layout done and general layout underway.
Research was done on hybrid promoters in the kit and two interesting ones were found. The genes coding for the necessary activators and repressors were also included in the distribution. The resulting four biobricks of interest <partinfo>BBa_R0062</partinfo>, <partinfo>BBa_K092400</partinfo>, <partinfo>BBa_C0078</partinfo> and <partinfo>BBa_C0079</partinfo>, were transformed using the standard protocol.
The BioBrick part <partinfo>BBa_K561001</partinfo> (vgb promoter, microaerobic) was extracted from the kit, transformed into E. coli, and the transformed cells plated out on Chloramphenicol (Cm) plates.
A transformed colony containing the RBS part extracted yesterday, was transferred to liquid medium (5 mL LB)
Two transformed colonies containing the DTT part on a plasmid backbone with Ampicillin and Kanamycin reistance, which was extracted yesterday, was transferred to liquid medium as follows:
One colony grown on agar plate with ampicillin, was transferred to a tube with LB (5 mL) + Kanamycin (100 ug/mL One colony grown on agar plate with kanamycin, was transferred to a tube with LB (5 mL) + Ampicillin (100 ug/mL)
DNA was isolated from the liquid culture of LuxR+HSL transformed cells inoculated yesterday by miniprep. The DNA concentration in the product was measured as 13,3 ng/uL.
The biobricks <partinfo>BBa_B0034</partinfo> and <partinfo>BBa_B0015</partinfo> were extracted from the distribution kit and transformed into E. coli which were plated out. The liquid culture left after the plating was stored at 5 C.
Plasmid DNA was isolated from liquid cultures, inoculated on 22.06 and 23.06 respectively, of E. coli transformed with <partinfo>BBa_R0062</partinfo> (LuxR & HSL promoter) and <partinfo>BBa_K112808</partinfo> (T4 lysis device, no promoter), using Promega miniprep. The concentration of isolated BBa_R0062 DNA was measured to 15,7 ng/uL. Due to the low yield, this brick may have to be regrown. Suspecting too long incubation time as the cause. The concentration of isolated BBa_K112808 DNA was measured to 111,0 ng/uL. Both samples were stored in the freezer at -20 C.
To make a new liquid culture of <partinfo>BBa_R0062</partinfo> transformed E. coli, a colony from the plate made on 21.06 (stored in refrigerator at 5 C since then) was inoculated in aproximately 4.5 mL LB medium with 4.5 uL of Amp stock solution (100 mg/mL) added, and incubated with shaking at 37 C.
Of two LA + Amp plates with <partinfo>BBaK112808</partinfo> transformants (20 and 200 uL) incubated since yesterday, only the 200 uL plate showed growth (10+ colonies). Negative control plate (untransformed cells) showed no growth. One colony was transferred to liquid culture.
On the 20 uL plate, a water bubble below the agar was mistaken for a colony, and a liquid culture was inoculated with a toothpick after scratching the plate. The mistake was realized, but the tube was still left to incubate, as it was thought it could act as a (weak) negative control.
The BBa_R0062 transformant from yesterday yielded several colonies on its plate, and one colony was inoculated into liquid medium and incubated at 37 C with shaking. After adding antibiotic to the liquid medium in the growth tube, some of the medium was spilled, so the growth volume was about 2 mL.
Isolation of plasmid DNA from all 7 initial transformants (see 20.06.12) was performed with the Promega Wizard Plus SV Minipreps DNA Purification System A1460.
The biobrick <partinfo>BBa_K112808</partinfo> (Enterobacteria phage T4 Lysis Device - no promoter) was extracted from the distribution kit and transformed into E. coli.
We started the day with a lecture on genetic circuit modelling by Ph.d student and iGEM team instructor Marius Eidsaa, followed by a discussion of the options available in designing our system.
All 7 transformants from yesterday yielded 1 or more colonies. Negative controls (untransformed cells) plated out on Kanamycin and Ampicillin plates showed no growth, indicating that the antibiotics were effective in selecting transformants for growth. 1 colony from each of the transformants was inoculated in liquid LB medium and incubated at 37 C with shaking.
The biobrick [http://partsregistry.org/Part:BBa_R0062 BBa_R0062] (Promoter (luxR & HSL regulated -- lux pR)) was extracted from the distribution kit and transformed using our modified version of the official iGEM transformation protocol (see Protocols).
We started the day with an introduction to computer modeling of biological sytems, and use of the Cain chemical kinetics simulation program. We then researched and discussed various biobricks, and transformed several biobricks from the iGEM DNA distribution plates into E coli.
The following biobricks were extracted from the iGEM 2012 DNA distribution kit and transformed according to protocol:
We started the day preparing lab equipment. We sterilized pipette tips, toothpicks, water and LA and LB medium. We also prepared stock solutions of ampicillin and kanamycin, and made petri dishes containing LA + Amp (100 ug/mL) and LA + Kan (100 ug/mL). Equipment and solutions were autoclaved at 120 C for 20 minutes.
We prepared LB medium (1 L) and LA medium (2 L). After mixing, each batch was divided into two bottles for autoclaving.
Stock solutions of Ampicillin and Kanamycin (both 100 mg/mL) were prepared from dry powders. After preparation and between use, the solutions were stored at -20.
After autoclaving, LA media bottles were left to cool. After reaching a temperature where they could be comfortably handled, the desired antibiotic was added using sterile technique. (500 uL stock solution to each bottle containing aproximiately 0.5 L, to a final antibiotic concentration of 100 ug/mL). The medium was then stirred and poured into petri dishes. The petri dished were left to cool down and placed cool for storage.
The team visited the lab and was given a EHS run-through by Merethe Christensen. We also risk evaluated the project and handed in the risk assessment, so now we are ready to start working in the lab:-)
Today, we tried to come up with a preliminary genetic circuit. We decided that using HGF as a signal molecule could be difficult, since we don't even know if a protein this size could penetrate the outer membrane and the peptidoglycan layer of E.coli. But we have found out that cancer cells excrete more lactate than healthy cells, so we decided to go for lactate, which is a small molecule. If the construct works, it could be modified to respond to other signal molecules.
Preliminary genetic construct:
We talked about what we had found out since last week. Rolf and Jarle had since last week investigated different ways of making a cell lyse, and they found out that genes for lysis are already in this years iGEM kit. Ove and Nina had been investigating toxins, but didn't find as much as they hoped for, but they have however found some toxins. Colisin E1 should be possible to use. They have also sent an email to Pål Fallnes, who Marit Otterlei suggested we could try to get in touch with. Apparently, he has been working quite a lot with expression of toxins in bacteria. Gunvor and Eirin have since last week been trying to find a suitable signal molecule we could detect. Marit Otterlei suggested that we could use HGF, so Eirin and Gunvor did some reasearch around this growth factor. They found out that HGF is the only known lignad to the receptor c-Met, which is a tyrosin kinase. The idea so far is to use this, find out what signaling pathways this receptor is connected to, and find the endpoint of the signaling pathway. We assume that the endpoint is a growth factor possibly regulating a promoter, and if we find such a promoter, this could be set to control the lysis genes. The problem is that we don't know how signals are transducted to bacteria from the exterior, and this proved to be hard to find any information about. But we know that proteins resembling tyrosin kinases called BY kinases exists in bacteria, and also that proteins from eucaryotic cells have sometimes been working in bacteria when introns are removed by using mRNA and reverse transcriptase.
Both in the case of toxins we can express, and a signal molecule we could get our cells to respond to; we need more time. So we decided that Ove and Nina will continue looking for toxins for another week, and Eirin and Gunvor will investigate signal molecules more in depth.
Rahmi have found some possibly useful biobricks, and will be sending references to the biobricks in question to the team by email. Rahmi had also baked a cake for today's meeting:-)
In the case of sponsors, many of the companies we have asked request a budget. Gunvor will try to get hold of this from Eivind, and she will also talk to Merethe Christensen, who is an engineer at dept. of biotechnology, to arrange a safety excursion in the lab, which is necessary before we're allowed to work there.
We also elected Nina as our photo chief:-)
We started continuing our dicussion on which toxins we should make our cells produce. If we manage to make the cells lyse only in the presence of the signaling molecule we chose to go with, it shouldn't mather what the cells produce, but if the system turns out to be leaky, it will be a problem if the toxins we prodce is too toxic. That way we would also kill healthy cells. Also, another problem with too toxic molecules is that we will need a special lab to work with them. So we decided to go for the happy mean. We also discussed the posiibility of making the intracellular concentration of toxins a checkpoint for lysis, so that even if a signal molecule is present, or the cells is experiencing an oxygen deficient environment, they would still not lyse until the concentration of anti cancer molecules is high enough.
We now have three different modules involved in our project; a production part, a detection part and a lysis part. We decided to split in three groups and investigate these modules in depth;
We also decided that we need a PR chief. Rolf volunteered!
We had our first meeting with our new advisor, Marit Otterlei, and we discussed which signal molecule we could use to detect the cancer cells in addition to using the O2 promoter to detect oxygen deficient areas. Prof. Otterlei suggested that we could use the Hepatocyte Growth Factor (abbreviated HGF), which is a growth factor regulating cell growth, cell motility and morphogenesis, that has the ability to bind to a tyrosine kinase. Prof. Otterlei also told us that many toxins can be produced by cells, so we decided to look more at making the cells produce toxins in addition to the ones we've already talked about.
We began this weeks meeting with going through the to-do-list from last meeting.
We discussed the possibility of using adenosine as a signal molecule, but Rhami was sceptical, as he thinks adenosine and ATP are taken up by the cells through passive transport. So that means we might have to look for another signal molecule. Since we have few ideas ourself, we decided to have a meeting early next week instead, and try to get our new advisor Marit Ottelei to attend the meeting, since she might have some ideas.
In the time leading up to the next meeting, Rolf will continue looking for sponsors, and the ones who has time should start looking at different ways to lyse a cell.
We had our weekly meeting. We started by going through what we have done since last week:
We also decided that we should start looking for sponsors. We will submit an application for funds to Programme of Bioinformatics (PBI), and Rolf and Jarle is going to contact VWR, Sigma-Aldrich, and Fisher Scientific.
Eivind reminded us that it is important to come up with an idea for what our genetic construct will actually look like as soon as possible, so we can start the modelling. We decided that we will decide on an idea for a genetic circuit on next meeting, which will be on friday 18.05, at 13:30.
Have a nice weekend:-)
I have been playing around with a wiki design scheme today which can be found here. I hope we can discuss the wiki design a little bit this friday. Also I have been trying to make a calendar solution, but I haven't found any easier or more user-friendly way to implent this than to simply use the default wiki setup. So, at least for now, I think we should just keep using this site the way it is and add updates the way Gunvor did below (and I am doing now) ;) When you have added a new post to a day, you can click the button "Your signature with timestamp" in the editing menu to add your username along with the current time and date.
Ove
We had a meeting, and we discussed several things we would like to look more into before we start planning what our genetic circuit will actually look like.
Here is a list of what we decided to do, and who will do it:
- We also decided to have our next meeting at 13:30 next friday, and we decided to eat lunch together on wednesdays
- Rahmi came with a suggestion for characterization of the O2-sensitive promoter:
Gunvor and Rahmi held an introductory lecture to cloning techniques. Gunvor held a crash course in molecular biology, the biobrick concept, and the most common molecular biology techniques, while Rahmi covered more advanced cloning techniques like SLIC and Gibson. If anyone wants more information on for example SLIC and Gibson, google j5 assembly;-)
Today, all the groups from last time started with giving an overview of possible project within their topic. Then we discussed for quite a long time, and in the end we decided we wanted to work with cancer. But we also decided to keep the biosynthesis of fatty acids as a side project. The electricity project turned out to be quite hard to complete in only two months, so we decided to drop it.
Our final project idea is then to make bacteria, for example E.coli cells, produce anti cancer drugs; preferably as many different molecules as possible. We have read about both enzymes and endpoints of metabolic pathways that are disadvantageous for cancer cells. Our engineered cells should also be able to respond to a signal molecule secreted in larger amounts from cancer cells than from normal cells, for example a signaling molecule that promotes angiogenesis. When the cells detect the signal molecule from the cancer cells, they should lyse, releasing the anti cancer molecule close to the tumour. Another approach to reach the tumour could be to take advantage of the fact that the environment inside tumours normally is oxygen deficient. And as E.coli cells naturally migrate towards areas low in oxygen, a possible solution is also to activate the lysis gene when the cells are in an are with little oxygen. Rahmi told us about a promoter that gets activated by low concentration of O2. This could regulate the lysis genes.
For the main project, we have to make the cells lyse at a certain concentration of an outer stimuli. For our side project it could be interresting to do the oposite; making the cells lyse when they have produced a certain amount of fatty acids.
Today, we decided on the overall top three projects for the team. We added the points all the team members have given to the different projects, and the list then becomes as follows:
| Project | Number of points |
|---|---|
| Cancer (search and destroy) | 14 points |
| Biosynthesis of fatty acids | 10 points |
| Bacteria as electrical switch | 6 points |
| Water purification - hormones | 3 points |
| Water purification - sucralose | 1 point |
| Oil spill removing bacteria | 1 point |
| Biomining | 1 point |
We decided to look deeper into the overall top three projects. We formed three groups of two and two, and decided to look at the following things for the next meeting:
The groups:
We also elected Ove as wiki chief:-)
Since we have quite a few project ideas to choose between now, we decided that for next meeting, all team members should pick their three favourite projects. We decided to give our favourite project three points, the project on second place two points, and our third favourite project one point. Before the next meeting we will add all the points together, and se which projects we should look at more in depth. At this meeting, we also discussed activities for Researchers Night. So far we have thought about making a construction kit to make the attendants on RN understand how we can combine different biobricks. We also discussed bringing pipettes, letting the attendants make alginate beads with different colours that they could bring home, and bringing a microscope and coloured cells (harmless, of course).
We kept discussing possible projects, and we now have several projects that would be interresting to work with. Here are the topics we have discussed so far:
We had a meeting, where we started discussing ideas for this year's iGEM project. We also decided to eat lunch together once a week, to get to know one another. For the outreach part of the project, Gunvor suggested that we could collaborate with Studentersamfundet to make a "lørdagsmøte" about synthetic biology.
We had our first meeting, and the team members met each other for the first time!
We recieved emails from Eivind letting us know that we are the ones that have been selected to represent NTNU in iGEM 2012. Everybody is happy!
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