iGEM 2021 — Team Siberia

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# Team:Siberia/Notebook

# Notebook

**Disclaimer:**The Notebook reflects mostly laboratory work and technical issues which can be easily formulated, and tends to ignore such tasks as communication/social network management/human practices etc, so if you want a detailed account on what exactly each team member has done please visit our [Attributions](https://2021.igem.org/Team%3ASiberia/Attributions)  page.

| Date | Event/Task | Participants | Results & conclusion |
| --- | --- | --- | --- |
| 13.05.21 | First meeting with the experts | Marina, Danil, Artemii, Alexander, Anastasia, Daria, Anna | Drop the idea of working with phages and Micobacterium smegmatis. |
| 25.05.21 | Second meeting with the experts | Marina, Danil, Artemii, Alexander, Anastasia, Daria | Formulating the Leap-2 producing bacteria as a project idea |
| 25.05.21-28.05.21 | Working on presentation for the sponsors | Marina, Danil, Artemii, Alexander, Anastasia, Daria, Anna | A presentation is made with clear project plan & description |
| June | Gathering literature, constructs design | Artemii, Marina, Alexander | Bacteria secretion systems overwiewed. Major goal for project: study the LMWP effect on the secretion. |
| 1.06.21-10.06.21 | Modeling of the putative Leap-2+Myc-tag+LMWP structure | Alexander | Protein model shows that domains does not interfere |
| June | Creating team project design and social network | Anna & Danil | Team Instagram and VK page are created |
| June | Establishing collaboration network | Daria | Meetups with teams |
|  | Team X meetup |  |  |
|  | Team Y meetup |  |  |
| 01.07.21-14.07.21 | Plasmid construct design | Marina, Alexander | Gene constructs and cloning strategy has been designed |
| 16.07.21 | Consultation with I.S. Dovydenko(Laboratory of Synthetic biology IChBFM SB RAS) on constructs design | Marina, Artemii, Alexander | Constructs synthesis has been ordered |
| 28.07.21 | First visit to the laboratory of Molecular Microbiology | Artemii, Marina, Uliana, Alexander | Getting to know the laboratory and the lab collective |
| 29.07.21 | Plasmid quality check | Marina | pBluescript plasmid is ok and linearized |
| 30.07.21 | pBluescript blunt ends ligation | Marina & Artemii | LIgation mix overnight incubation |
| 02.08.21 | E.coli Top10 transformation | Artemii & Uliana | 10 transformations have been done |
| 03.08.21 | PCR colony | Artemii | Insertion is present, orientation is unknown |
| 04.08.21 | PCR colony | Artemii | Orientation is verified, but wrong one |
| 05.08.21 | PCR colony | Artemii | 7 of 10 correct insertions have been found |
| 06.08.21 | Plasmid DNA extraction | Marina | Success |
| 09.08.21 | pSG4K5 linearization | Marina | Success |
| 10.08.21 | Sequincing request, PCR colony | Artemii | 1 more successive transformant found |
| 11.08.21 | Transformation | Artemii & Uliana | Success |
| 12.08.21 | PCR colony | Artemii & Uliana | 8 positive colonies. Sequincing results allows for chosing transformants with no mistakes |
| 13.08.21 | pDNA extraction | Marina | Success |
| 16.08.21 | Cutting the inserts out of pBluescript | Marina | Success but very low concentration |
| 17.08.21 | Ligation | Artemii | Ligation mix is made |
| 18.08.21 | BL21 transformation | Artemii & Uliana | Rare colonies |
| 19.08.21 | PCR colony | Artemii & Uliana | All negative. 300 and 400 PCR bands everywhere, what does it mean? |
| 21.08.21-28.08.21 | August synthetic bilology school | Marina, Uliana, Artemii, Alexander, Anna, Anastasia | Almost whole team got involved into the school organization |
| 23.08.21-26.08.21 | Top10 transformation with pSG4K5 | MArina, Artemii, Uliana | WRONG PLASMID (first transformation attempt)! And again those bands |
| 27.08.21 | Top10 transformation with pBluescript to get more DNA | Artemii | Success |
| 30.08.21 | pDNA extraction | Anastasia | Low DNA yield |
| 31.08.21 | Changing the cloning strategy: PFU PCR to add an Spe restriction site to the original synthesised genes | Artemii | All bands are correct |
| 01.09.21 | Restriction and ligation (plasmid as control) | Artemii | Plasmid is ok means everything is ok? Spoiler - no |
| 02.09.21 | Bl21 transformation | Uliana | Success? |
| 03.08.21 | PCR colony | Artemii | Nothing. Maybe, we should try Top10? |
| 06.09.21 | Top10 transformation | Uliana | Ok |
| 07.08.21 | Colony PCR | Artemii | Nothing. Prof. N.V. Tikunova says the problem might be due to gel extraction kit inhibits the later enzymatic reactions |
| 08.09.21 | PFU PCR | Artemii | More DNA needed |
| 10.10.21 | Restriction & ligation. DNA precipitation to overcome inhibition and gel electrophoresis to verify the restriction | Artemii | Not all genes have been cut |
| 13.09.21 | Top10 transformation | Artemii | Ok |
| 14.09.21 | PCR colony | Artemii | Strange bands again. p111 positive? p111 and p211 (with wierd bands) taken for DNA extraction |
| 15.09.21 | DNA extraction and PCR colony | Marina & Uliana | Low DNA yield again. 211 didn't grown. No more positive colonies |
| 16.09.21 | Ligation (now also precipitate the DNA before the ligation) | Artemii | Ok |
| 17.09.21 | Plsamid clearance for sequincing (extracted 15/09/21) | Artemii | not enough plasmid |
| 22.09.21 | NEB competent cells 10b and DH5a transformation | Marina | Not everything has grown. |
| 06.10.21 | PCR colony | Uliana | PCR shows no positive results but weird bands at 300 bp and 400 bp |
| 08.10.21 | Overnight cultures for plasmid extraction | Marina | ok |
| 09.10.21 | Restriction analysis with easy miniprep | Marina | Uncertain results |
| 10.10.21 | Analysis repeat with more DNA | Marina | 2 postive colonies? Could PCR colony be wrong? |
| 11.10.21 | DH5a transformation with 16.09 ligations to get more DNA | Marina | ok |

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# Team:Siberia/Engineering

LEAP2BRAIN

[![IGEM Siberia](https://static.igem.org/mediawiki/2021/8/8d/T--Siberia--logo.png)](https://2021.igem.org/Team%3ASiberia)

[![](https://static.igem.org/mediawiki/2021/3/3d/T--Siberia--logoblack.png)](https://2021.igem.org/Team%3ASiberia)

### [Home](https://2021.igem.org/Team%3ASiberia)

### Project

- [Description](https://2021.igem.org/Team%3ASiberia/Description)
- [Engineering](https://2021.igem.org/Team%3ASiberia/Engineering)
- [Models](https://2021.igem.org/Team%3ASiberia/Model)
- [Experiments](https://2021.igem.org/Team%3ASiberia/Experiments)
- [Safety](https://2021.igem.org/Team%3ASiberia/Safety)
- [Results](https://2021.igem.org/Team%3ASiberia/Results)
- [Contribution](https://2021.igem.org/Team%3ASiberia/Contribution)

### [Human Practices](#link2)

- [Human Practices](https://2021.igem.org/Team%3ASiberia/Human_Practices)
- [Education & Public Engagemet](https://2021.igem.org/Team%3ASiberia/Communication)
- [Best Education](https://2021.igem.org/Team%3ASiberia/Education)
- [Implementation](https://2021.igem.org/Team%3ASiberia/Implementation)

### [Team](#link2)

- [Collaborations](https://2021.igem.org/Team%3ASiberia/Collaborations)
- [Attributions](https://2021.igem.org/Team%3ASiberia/Attributions)

Leap2Brain
Siberia

# Engineering Success

### A novel delivery system for the treatment of obesity

### **Intermediate vector successful cloning**

The most significant our results in the lab. Let us show you our first cloning strategy:

![](https://static.igem.org/mediawiki/2021/e/ea/T--Siberia--Cloning_strategy1.png)

We have successfully cloned all 10 constructions: coding YebF with and without LMWP and 4 pairs with different secretion tags. We needed YebF to study how LMWP would influence the bacterial secretion, for YebF is known to be secreted by E. coli K12. The results were further verified by sequencing – it showed no critical mistakes. However, we then faced a problem with extracting enough DNA for the restriction digestion and gel purification. It was just the moment when our collaborators came up with the idea of using high fidelity PCR to obtain our insertions in required concentrations. Great idea, but did it mean, that all we had done before was in vain? One may say: “That intermediate vector cloning was a laborious mistake, you shouldn’t have done it!”. But look at benefits we gained from it:

We have successfully cloned all 10 constructions: coding YebF with and without LMWP and 4 pairs with different secretion tags. We needed YebF to study how LMWP would influence the bacterial secretion, for YebF is known to be secreted by E. coli K12. The results were further verified by sequencing – it showed no critical mistakes. However, we then faced a problem with extracting enough DNA for the restriction digestion and gel purification. It was just the moment when our collaborators came up with the idea of using high fidelity PCR to obtain our insertions in required concentrations. Great idea, but did it mean, that all we had done before was in vain? One may say: “That intermediate vector cloning was a laborious mistake, you shouldn’t have done it!”. But look at **benefits** we gained from it:

- The competent cells preparation protocol verified
- Transformation skills improvement
- Matrix with no mistakes obtained for the high fidelity PCR

First two points helped us later while we were looking for possible mistakes in the transformation process

![](https://static.igem.org/mediawiki/2021/0/00/T--Siberia--pBluescript_gel_image_blunt_ends_ligation.tif)

A successful colony PCR result. The number here is a colony code, and the length is given for those bands, that has the expected length

![](https://static.igem.org/mediawiki/2021/d/d5/T--Siberia--PFU_PCR.png)

High fidelity PCR results with all constructs having expected length

### **Successful troubleshooting after receiving bad transformation results but failing to obtain final construct**

Let’s look at our new cloning strategy:

![](https://static.igem.org/mediawiki/2021/e/e2/T--Siberia--Cloning_strategy2.png)

Cloning strategy #2

What problem did we face here? Each transformation resulted in zero positive results (PCR colony), but weird bands at 300 and 400 bp. What was our troubleshooting pathway?

![](https://static.igem.org/mediawiki/2021/3/3b/T--Siberia--Weird_bands.jpg)

Weird bands of unexpected length

1. Do a negative control PCR with the original plasmid (no weird bands)
2. Try in silico PCR to find possible additional sites for primer binding in plasmid with target insertions (no such sites found)
3. Try fresh antibiotic solution
4. Try Top10 strain instead of BL21, which is hard to transform
5. Check the restriction: compare cut and uncut gene fragments on gel electrophoresis.

Surprisingly, we have discovered the length was just the same, which meant
that restriction had failed. But we had performed a control for both of the enzymes.
5 U each was sufficient to linearize 1 μg of pSG4K5 vector.
The problem was somehow connected to our gene constructs. Prof. N. V. Tikunova advised us
to do ethanol precipitation of the genes DNA – she said, that the kit, we had used to purify the DNA
after the PCR might inhibit further enzymatic reactions. The effect was due to residual cations from the spin column membrane,
she explained. We did the precipitation and saw the fragments were cut!

![](https://static.igem.org/mediawiki/2021/b/ba/T--Siberia--Successful_restriction.jpg)

Unfortunately, it was not the only reason of transformation problems: after doing the transformation once again we still received those weird bands. For now, we have not enough time to properly understand them, but here, is the list of possible **explanations:**

1. Primers have binding sites somewhere in the E. coli genome.
2. Leap-2 or LMWP is toxic for E. coli and transformed bacteria cannot survive.
3. A kind of recombination occurs with our plasmid.
4. Some ligation issues.

**And the list of actions to be taken to understand what has happened:**

1. 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.
2. Extract those bands source whatever it is and get a sequence of it. Thus, if they occur due to recombination – we will see it.
3. 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.
4. 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.

It seems we still have plenty of work to do, however we already have a plan for further troubleshooting. And we are not going to stop in the middle.

### Theoretical success: a detailed in vitro model design

**Original plans**

As our project is a proof-of-concept, combining the idea of living therapeutics and nose-to-brain delivery of macromolecules, ideally we need to model and test both of these aspects. Originally, provided we demonstrated secretion of the LEAP2 fusion protein from bacteria, we were planning to move straight onto in vivo murine models to measure the success of N2B delivery using immunohistochemical techniques. Needless to say, at the time we were also concerned with the ethical aspect.

For a more comprehensive analysis, we would have first tested the delivery of the purified fusion protein, using a protocol such as the one used by Hansen et. al., 2013 [1]. Only under the condition that this step worked, would we have considered moving on to actually planting the modified bacterium into the nose (ideally not the E.Coli we used to demonstrate the secretion of our protein, but a native bacterium found in the microbiota of the nose (i.e. Neisseria spp). Obviously, the second step was unrealistic for the timeframe of the competition, however, should the concept of combining living therapeutics and N2B delivery continue its development in the real world of science, unfortunately, animal testing would be necessary at some point to test for the safety of our treatment. In fact, our Human Practices survey showed that the safety of our innovative therapy was the main area of interest for both scientists and average citizens.

**What changed?**

In one of our Zoom calls with team CityU, one of their team mentioned that they’ve found an in vitro model of the blood-brain barrier to use in their project. This prompted us to also step away from in vivo experiments completely for the time being, and instead, look into researching and designing an in vitro model of the mucosal barrier in the nasal epithelium, suitable for our goals.

Almost immediately we ran into some challenges. We haven’t found an existing model of the nasal mucosal barrier that will account for all of the physiological properties of the nasal epithelium that might affect nose-to-brain delivery. Most standard models [2] that use an air-liquid interface Transwell ®/Franz cell system with a synthetic mucosal layer and standard RPM 2650 and Calu-3 respiratory cell lines do not account for the olfactory or trigeminal epithelium in the nose, which is crucial for our goals (as the delivery of the protein to the brain happens via the trigeminal and olfactory neurons). The closest model we found to that is one that uses nasal olfactory epithelial cell lines by Ladel et al., 2019 [3]. One idea we had was to combine this model with one by Charles et al. [4] which supported the colonization of Transwell ® systems by nasal microbiota, thus allowing us to test both the penetration of the mucosal barrier by the protein and its uptake by the cell monolayer. The main area of concern was how accurately this would model the olfactory epithelium. More accurate organ-on-a-chip models, such as the one by Na et al. [5], which includes the gland-like structures that secrete the mucus,
do exist but are overly complex for our goals and a subject of research in their own right.

Though the initial concept was quite simple, the lack of existing models meant that we would have to design our own. As this was somewhat out of our depth, we decided to consult on this matter with an expert in N2B delivery and in vitro modelling of the nasal epithelium, Dr. Romashchenko of IC&G SB RAS. A key insight that he left us with is that technically, we don’t even need to model the olfactory epithelium itself - the nasal microbiota tends to reside in the sinuses, where cells don’t have many cilia and the mucosal current isn’t so strong so as to completely wash away our product before it even penetrates the mucosal barrier, as is the case for the olfactory epithelium. In fact, the secreted product would travel from the sinuses and wash over the olfactory bulbs together with the mucus. This immensely simplified our task.

**Current plans**

The article, on which we based our idea of making a fusion protein with the cell-penetrating peptide LWMP [6] states that the mechanism by which their fusion protein travels to the hypothalamus via the olfactory and trigeminal nerves is unclear. It is speculated that endocytosis and macropinocytosis play an important role, however, the intra-tissue penetration mechanisms are “largely unknown” and most likely occur via an extracellular mechanism.

Therefore, uncovering the underlying mechanism behind this type of delivery is a point of interest to us, and an interesting area of research in itself. To account for both potential mechanisms, we decided to use two simple in vitro models.

The first model would test for paracellular penetration via “leaky” tight junctions and would be a simple ALI monolayer system, similar to the ones we were originally researching [7]. The cell type isn’t crucial, provided the cells in the monolayer form tight junctions. In this model, we would also test the interaction of our protein with mucins in an artificial mucosal layer.

![](https://static.igem.org/mediawiki/2021/a/a3/T--Siberia--Paracellular_transoprt_scheme.jpg)

An in vitro model for paracellular transport in monolayers scheme

The second model would test for intracellular transport in neurons. We plan to use a microfluidic model to remove the effects of diffusion, similar to the one shown in the image [8]. In the neurons, we would monitor the localization of LEAP (i.e. in lysosomes, the Golgi apparatus, the ER). We can do this with the help of the Myc-tag found in our protein via immunostaining or replace the Myc-tag with a fluorescent tag. In the case of intracellular mechanisms of transport, we would ideally, find our protein in synaptic vesicles and demonstrate synaptic transmission.

![](https://static.igem.org/mediawiki/2021/b/bf/T--Siberia--Neuron_transoprt_scheme.jpg)

An in vitro model for intracellular transport in neurons scheme

For both models, suitable cell lines are available at IC&G SB RAS and ICBFM SB RAS, and we plan to use those of the required safety level and double-check with the Safety and Security Committee at iGEM prior to doing any after iGEM lab experiments.

As a final point, though in vitro models have many advantages, even the most complex ones cannot account for all of the interconnected physiological effects happening in the organism. Our models, for example, do not take into account the effect of gravity - the olfactory epithelium the mucus washes over is “upside-down”. Nor do these models account for the fact that the volume of the olfactory bulb can fluctuate in response to changes in the sense of smell [9]. On ethical grounds, an additional step may be added after our in vitro models and prior to doing any fully-fledged in vivo tests on mice - a model using murine olfactory epithelium slices, for example. In any case, more complex experiments such as these do fit in our vision of the implementation of our project, they cannot be realistically
done in the timeframe of the competition and we consider them to be areas of future research and further development.

**References**

1. Hanson, L. R., Fine, J. M., Svitak, A. L., & Faltesek, K. A. (2013). Intranasal administration of CNS therapeutics to awake mice. Journal of visualized experiments : JoVE, (74), 4440. https://doi.org/10.3791/4440
2. Boegh, M. & Foged, Camilla & Müllertz, Anette & Nielsen, Hanne. (2013). Mucosal drug delivery: Barriers, in vitro models and formulation strategies. Journal of Drug Delivery Science and Technology. 23. 383. 10.1016/S1773-2247(13)50055-4.
3. Ladel, Simone, et al."Improved in vitro model for intranasal mucosal drug delivery: primary olfactory and respiratory epithelial cells compared with the permanent nasal cell line RPMI 2650." Pharmaceutics 11.8 (2019): 367.
4. Charles, D. D., Fisher, J. R., Hoskinson, S. M., Medina-Colorado, A. A., Shen, Y. C., Chaaban, M. R., Widen, S. G., Eaves-Pyles, T. D., Maxwell, C. A., Miller, A. L., Popov, V. L., & Pyles, R. B. (2019). Development of a novel ex vivo nasal epithelial cell model supporting colonization with human nasal microbiota. Frontiers in Cellular and Infection Microbiology, 9(MAY), [165]. https://doi.org/10.3389/fcimb.2019.00165
5. Na K, Lee M, Shin HW, Chung S. In vitro nasal mucosa gland-like structure formation on a chip. Lab Chip. 2017 May 2;17(9):1578-1584. doi: 10.1039/c6lc01564f. PMID: 28379223.
6. Lin, Tingting et al. “Nose-to-brain delivery of macromolecules mediated by cell-penetrating peptides.” Acta pharmaceutica Sinica. B vol. 6,4 (2016): 352-8. doi:10.1016/j.apsb.2016.04.001
7. Lechanteur A, das Neves J, Sarmento B. The role of mucus in cell-based models used to screen mucosal drug delivery. Adv Drug Deliv Rev. 2018 Jan 15;124:50-63. doi: 10.1016/j.addr.2017.07.019. Epub 2017 Jul 25. PMID: 28751201.
8. Martins, L.F., Costa, R.O., Pedro, J.R. et al. Mesenchymal stem cells secretome-induced axonal outgrowth is mediated by BDNF. Sci Rep 7, 4153 (2017). https://doi.org/10.1038/s41598-017-03592-1
9. Negoias S, Pietsch K, Hummel T. Changes in olfactory bulb volume following lateralized olfactory training. Brain Imaging Behav. 2017 Aug;11(4):998-1005. doi: 10.1007/s11682-016-9567-9. PMID: 27448159

LEAP2BRAIN

SIBIRIA

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# Team:Siberia/Experiments

# Protocols

Here are not only protocols, but tips we consider being useful for the lab work. They may help the beginners, but even experienced people could find some of them interesting

### Agarose gel electrophoresis (non-sterile conditions)

1. Measure 0.5 g (samples > 1000 bp), or 0.75g (200 bp < samples <1000 bp) of agarose
2. Prepare at least 350 ul of 1x TAE with Ethidium Bromide at final conc ~ 0.3μg/mL (MUTAGEN! WEAR GLOVES AND LAB COAT)
Tip: It will save your time, if you prepare larger volume of 1x TAE with Ethidium Bromide once and than will use it3. Mix it with 50 ml 1x TAE and boil until agarose is melted.
4. Let the gel cool down to 50 °C, until you'll feel comfortable to touch it with your hand
5. Pour the agarose into a gel tray with the well comb in place. Wait until it is completely solid (~ 20 min at room temp)
6. Place the gel into the gel box and cover it with 1x TAE from step 2
7. Carefully load your samples into the wells. Mix it with loading buffer if needed
Tip: You can use the inner sterile side of the Parafilm slide to mix samples with buffer8. Turn on the voltage. You can vary the parameters to achieve better speed or resolution but the defaults are 10d V, 200 mA, where d is the distance between the electrodes
9. Check the migration rate watching the loading buffer
10. After the process ends visualize the DNA/RNA using any source of UV-light (wear glasses)

### Ethanol DNA precipitation (non-sterile conditions)

1. Add 0.1 V of 3-5 M NaCL or NaOAc to your sample
2. Add 2.5-3 V of 96% ethanol
3. Vortex and put at - 80 °C for 30 minutes
4. Centrifuge 13 kg, 10 min, 4 °C (temperature influences the DNA amount greatly)
5. Carefully remove **ALL** the supernatant (don't disturb the residue, which could be almost invisible)
6. Let the residual ethanol to evaporate incubating samples in open tubes at 5 min, 37 °C
7. Dilute your DNA in sterile water (mQ if needed) and store at - 20 °C
Tip: The protocol will help to clear your DNA from salts, prepare it for the electroporation, increase the concentration etc. But it will reduce the total amount of the DNA

### Blunt ends ligation tips (non-sterile conditions)

1. Recommended vector/insertion molar ratio for the process is 1:50
2. Vector **dephosphorylation** is highly recommended prior to the ligation. Note, that alkaline phosphatase can show exonuclease activity, if incubated more than written in manufacturer's recommendations
3. Insertion could be additionally treated with **T4 polynucleotide kinase** prior to the ligation, for the further increase of the success probability
4. Don't forget to deactivate the ligase prior to the transformation. 10 min at 65 °C is enough for the T4 DNA ligase. Active ligase can hinder the transformation
5. To verify the correct orientation with the PCR colony use the vector-specific forward primer and the insertion-specific reverse one

### Preparing reagents for the effective heat-shock transformation (non-sterile conditions)

| Reagent name | Components |
| --- | --- |
| MOPS I | 1. 0.1045 g of 3-(N-morpholino)propanesulfonic acid 2. 0.0603 g of RbCl 3. 50 ml of dH2O |
| MOPS II | 1. 1.045 g of 3-(N-morpholino)propanesulfonic acid 2. 0.0603 g of RbCL 3. 2.5 ml of 1M CaCl2 4. 50 ml of dH2O |

Filter MOPS reagents through a 0.22 μm filter prior to use and store at 4 ° C.

### Competent cells preparation & heat shock transformation (sterile conditions)

1. Add 50\***N** μl overnight bacteria suspension to **N+1** ml of LB-medium, where **N** stands for the number of transformations your are going to perform
2. Incubate with shaking at 37 °C for 2h, then 20 minutes on ice
3. Transfer the bacteria to **N** 1.5 ml Eppendorf tubes.
4. Centrifuge 1 ml of bacterial suspension at 6000 rpm for 4 min at 4 °C
5. Remove the supernatant
6. **Gently** resuspend the bacteria in 1 ml of MOPS I
7. Repeat the steps 4
8. **Gently** resuspend the bacteria in 1 ml of MOPS II
9. Incubate for 15 min on ice
10. Repeat the step 4
11. Resuspend cells in 100 μl of MOPS II
12. Add the DNA (a total of 50 ng will be optimal)
13. Incubate 30 min on ice
14. Heat-shock at 42 °C for 2 min
15. Add 900 ul of LB medium to each tube
16. Incubate 1 h at 37 °C. While incubating prepare Petri dishes with LB agar and the desired selective markers
17. If the DNA you have added at the 12th step was a plasmid, extracted from bacteria, you should only plant 50 μl of bacteria suspension
18. If the DNA was the result of ligation, plant all cells: centrifuge them at 6000 rpm for 2 minutes and remove 900 μl of supernatant. Plant all bacteria resuspending them with the remaining 100 μl
19. Incubate the Petri dishes at 37 °C for 14-16 h

### Colony PCR (sterile conditions)

1. Preapare an 0.5 μl Eppendorf tube for each colony you are going to check
2. Add 20 μl of sterile water to each tube
3. With a sterile toothpick/pipette tip grab one half of the colony of interest and put it into the water
4. Heat the tubes at 95 °C for 10 min, then centrifuge at 13000 g for 5 minutes
5. Use the supernatant as matrix for the PCR reaction with the insertion specific pair of primers

# Kits and reagents

Here are all the kits and reagents which we have used according to the manufacturers protocol

| Name | Purpose | Notes (+, if works as expected) |
| --- | --- | --- |
| Biolabmix LR HS-ПЦР-Color (2×) | High-fidelity PCR | + |
| Sibenzyme Taq-pol with standard buffer | Colony PCR | + |
| Evrogen Cleanup Standard gel purification kit | Clear the DNA after PCR, restriction digestion, or agarose gel | DNA ethanol precepitation is required prior to the restriction, becuse inhibition may occur |
| Biolabmix Plasmid DNA extraction kit | Plasmid DNA extraction | Low yield |
| Sibenzyme T4 DNA ligase | Ligation | + |
| Sibenzyme Restriction enzymes: PciSI, AhlI, EcoRV | Restriction digestion of the DNA | PciSI and AhlI can't be heat-inactivated (manufacturer's information) |

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Siberia/Results

# Results

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.

![](https://static.igem.org/mediawiki/2021/6/68/T--Siberia--ASBS_All_participants.png)

ASBS participants and organizators

### Summary

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.