It is the first solo project for our team and the first real research where we decided our experimental path on our own. Here is the list of positive results of all our decisions, but some of the steps later turned out to be not really effective.
- Intermediate vector successful cloning & obtaining gene constructs
- Successful troubleshooting after receiving bad transformation results
- Finding an alternative for external markers for living therapeutics design
- Finding an alternative for mice tests
- Organization of the August Synthetic biology school
There were, however negative ones:
- Mistakes in cloning strategy
- Failing to obtain final construct despite numerous attempts and successful troubleshooting events
- Failing to show how LMWP influence the bacterial secretion
Let’s go into details of some points to see the work our team has gone through.
Intermediate vector successful cloning & obtaining gene constructs
We succeeded in obtaining ten pBluescript SK (-) vectors with 10 different insertion constructs. However, there were a problem with extracting enough DNA, so we decided to change the strategy and PCR the constructs out of plasmid with the required restriction sites. In brief, benefits:
- The competent cells preparation protocol verified
- Transformation skills improvement
- Matrix with no mistakes obtained for the high fidelity PCR
The only minus is a kind of waste of time. You can read more about the result achievement at our engineering page.
Successful troubleshooting after receiving bad transformation results
During the trouble shooting we have successfully found the source of one major problem: it was the kit, which inhibited the restriction digestion. However, it turned out to be not the last bug, so we didn’t manage to get an expression construction. You can read the full debug report at our Engineering page.
Possible other sources of trouble:
- Primers have binding sites somewhere in the E. coli genome.
- Leap-2 or LMWP is toxic for E. coli and transformed bacteria cannot survive.
- A kind of recombination occurs with our plasmid.
- Some ligation issues.
Suggestions for further troubleshooting:
- Order primers specific to the pSG4K5, which have already been tested by the plasmid designer. So we will be sure in their specificity, and probably will stop receiving those dismoraling weird bands after the PCR colony.
- Extract those bands source whatever it is and get a sequence of it. Thus, if they occur due to recombination – we will see it.
- Check the ligation with PCR by taking forward primer specific to pSG4K5 and reverse one – specific to the insertion. Thus we will see if there any correct insertions in the mix.
- To test toxicity, we will need an inducible promotor. Luckily, we already have our constructs cloned in the MCS of pBluescript vector. We can cut them and clone in an appropriate vector with an inducible promotor and see, if bacteria will die after the induction.
Finding an alternative for external markers for living therapeutics design
Building up a regulatory pathway is extremely important for the living therapeutics design. But the choice of meal-connected signals in the nasal cavity is extremely poor. We consider a great result of our brainstorming, that we have discovered an alternative: why do we need an outer signal, if we can just help people stick to their diets? We are going to make our bacteria to secrete Leap-2 periodically, increasing the appetite when it is dinner or breakfast, and decreasing it after the meal is taken to prevent uncontrolled snacking.
As long as we know it is the first living therapeutics project exploiting the genetic oscillator for the regulation. We believe there would be more of them, because circadian rhythms play crucial role in our health, so why not use them? You can read more about this at our Model page.
Finding an alternative for mice tests
The longer worked on our project the less we liked the idea of mice testing. A need for an intermediate stage between the protein production in culture medium and in a murine nose was obvious for us. Thus we have developed an experiment which can answer many questions before the animal execution. It will allow us tune our project better while in vitro and save animal lives. The experimental design is based on literature and expert’s consultations. You can read more about this at our Engineering page.
Organization of the August Synthetic biology school
TOur team has taken part in organization of the August Synthetic biology school. We were laboratory assistants, had a lecture about how to participate in iGEM, arranged hotel bookings etc. It has been a pretty busy week that we are proud of! You can read more about this at our Education page.
We have done quite a job working on our project. Despite certain misfortunes we are eager to comprehend the problems source and finish the job. We already have plans for future lab work troubleshooting. We also believe, that some of our findings and ideas will inspire future iGEM teams and help them design their projects and avoid some mistakes.