Team:UANL Mty-Mexico/project/DNA-Program-Delivery

DNA Delivery System

What is a DNA delivery system?
A DNA delivery system is a method by which exogenous DNA is introduced into an organism. These systems can be divided into two general categories: viral delivery systems and the non viral delivery systems. The viral delivery systems are the ones that use the viral infection mechanism to deliver genetic material, usually in a viral particle incapable of replicating itself. On the other hand, the non viral delivery system comprehends a bigger set of strategies:

• ● Electroporation, which is the introduction of DNA into a cell without a wall using an electric field to form pores in the membrane.
• ● Microinjection is another mechanism which consists in introducing DNA by pressure into an isobaric system.
• ● Lipofection as well is a strategy that introduces genetic material by using particles known as liposomes.
• ● Among others.

DNA Delivery Systems on Eukaryotes

With evidence on induced Pluripotent Stem Cells (iPS cells).

Justification

Cell reprogramming for generation of iPS cells is a technique which uses transduction systems with “reprogrammation factors”, proteins and transcriptions factors involved primarily in the methylation patterns in the genome of a differentiated cell, which are modified in order to produce a cell capable of differentiating again, into different types of cells, but not a whole organism. While this reprogrammation might seem different from the “biohacker” system we propose, it is the same basic principle; the use of a gene/DNA delivery system, in this case, for a mammalian cell; the uses of this biohacker system with this type of DNA delivery system will be discussed, while reprogrammation efficiency cannot be discussed, since it relies on more complex factors, as the complete modification of methylation patterns, the stability of the new methylation patterns and a steady state of other differentiation and growth factors.

Retroviruses.

A common tool, used in both clinical gene therapy and basic research, are retroviruses; their biology is well understood, and they have a high efficiency on both transduction and expression of genes, and can be replication-competent (it has in its genome the essential genes for virion synthesis) and replication defective; thus, the technique is widely used. Nevertheless, the virus genome is large, so it has a limited cloning capacity for multiple genes, which makes a reprogrammation difficult on whole systems or modules on synthetic biology or induction of pluripotent cells.
Even though, in the work of Yamanaka, the first delivery of four reprogrammation factors on rat fibroblasts in order to make iPS was made using a Moloney murine leukemia virus (MMLV) based retrovirus vector, but this system often reactivated the MMLVLTR promoter, causing tumors due to expression of c-Myc factor; when this promoter was removed, they got completely functional iPS cells.
Better results have been reported with other strains of cells, reprogrammation factors or retroviruses.

Lentiviruses.

In contrast with retroviruses, lentiviral systems can infect both dividing and non-dividing cells, whilst retroviral systems can only infect dividing cells. All other features are shared, including retrotranscription and genome integration.
Though lentiviral systems integration is unpredictable, it is less likely that they integrate in oncogenic regions of the genome, causing cancer, than gamma-retroviral systems. Unlike the retroviral systems, all of the lentiviral systems have been designed replication-defective.

Episomal vectors

Most plasmids cannot replicate themselves in a mammalian environment, therefore, they only express transiently. Nevertheless, plasmids like oriP/Epstein-Barr nuclear antigen-1 replicate autonomously as extrachromosomal elements without integration in cells, dividing or non-dividing.

Adenovirus

Adenoviral systems are also non-integrative vectors (except on eggs), can transfect both replicative and non-replicative systems, as well as almost every kind of cell, except for certain lymphoid cells.

Others

In iPS cells, excisable integrated vectors are also used in order to generate iPS transgene free cells, capable of differentiating, but the biohacker system itself does not consider the excision of the transgenes at any moment, it rather focuses on the introduction of new programs in the same cell, as a way of direct “differentiation”, so excisable integrated vectors were not considered.

Animal-bacteria horizontal gene transfer.

Horizontal gene transfer has been described in a wide variety of organisms, mostly prokaryotes; whole genome analyses show that genes are transferred horizontally between closely related taxa, and between bacteria inhabiting the same environment.
Horizontal gene transfer is also found in eukaryotes, and even on superior animals; examples range from the transfer of P elements between Drosophila melanogaster and D. willistoni, the transfer of genes for carotenoid biosynthesis from fungi to pea aphids and the more recently discovered heritable lateral gene transfer in humans and Trypanosoma cruzi.
Although rare, interkingdom gene transfer has also been observed, the Eubacteria Thermotoga maritime has 81 archeal genes, and the well known Eubacteria-Eukaryota transference; Agrobacterium tumefaciens transfers 10-30kbp to plants. In the case of bacteria to animals, it happens through symbiosis, like the symbiotic relationship between Wolbachia with arthropods and filarial nematodes.
Though not a DNA delivery system in itself, the danger of transferring the program to other species, bacteria, animals or plants, has to be considered in function of the organism we are using.

Description - Objective - Explanation - Bases - Conclusion

Synthetic Rally

Description - Return

One of our activities for human practices consisted in creating a rally that would teach synthetic biology to students from 9th grade. We wanted to show something that was, in some cases, difficult to explain. The plan was to teach them some basic things about molecular biology that would help them understand each of the bases of the rally, including genes and biobricks, and how they could use these to design a circuit for a project. We decided that each of the bases would represent different parts of this year’s circuit, explaining how each of the parts work. For example, base one represented the Promoter; base two, the Riboswitch, and so on.

Objective - Return

The objective was mainly to help students understand that genetically modified machines work, basically, like a normal machine. We wanted to explain each of the parts that formed our circuit, how each of them worked according to its function, and how every part is necessary for the circuit to work. But all of these are hard to understand when you can’t see the relation between both types of machines. When you tell people who know nothing about genetic engineering that you are building a genetic machine, they have no idea of what to imagine; of how that could work. So we tried to show it to the students in the simplest way.

Explanation - Return

The first thing we did when arriving to the school, was to organize all of the materials needed for each one of the bases. While we were doing this, three of our team members went to one of the classrooms that the school had lent us, and explained some basic things about DNA and genes to the students. They explained it in a simple manner, just so the students could understand that these were needed to form the biobricks, and these last ones to form a circuit. But that part was explained later on.

After finishing the presentation, the 40 students from the first classroom went outside, to the first base. We divided them in two groups, and gave the first twenty of them small ribands of different colors, to divide them in teams for them to compete. The other twenty were kept waiting, while some of us asked them questions for a survey about transgenic food.

We handed out one small card with the drawing of our circuit to each one of the teams from the first group; a card that we would mark, whenever they finished one of the bases, with the points they had earned. The one who came out first place would receive 4 points; second place would receive 3, and third and fourth place would receive 2. We had one person in charge of writing down the points in each card to keep track of who was winning. After explaining all of this, the rally began.

The first thing we did at each one of the bases was to identify the name; say if it was the promoter, the terminator, the RBS… and we then gave an explanation of what it was without explaining completely how it worked. At the end of the activity, we would state the relationship between what they had played and the way the actual part of the circuit functioned. Then the one in charged would yell, and they would change bases. When the first group passed to the second base, the group that was kept waiting entered the first base.

Bases - Return

''BASE 1: PROMOTER'' - The activity was called Streets and Avenues, but the game was changed a little bit. All the teams would be aligned, forming a square, and one person from each team would be standing at each corner. That person would have to find his/her match; someone with the same color inside the square. For example, maybe someone form team Blue, inside the square, would be Yellow. The Yellow person outside would have to find that Blue person before the others found their match, with the path changing just as it does in the common “Streets and Avenues“ game.

''How does this relate to the promoter?'' The promoter needs to find the one thing that starts it, for the circuit to start too.

''BASE 2: RIBOSWITCH'' - This activity was called Dragons. Each team had to form a line and hold hands, being really careful not to let go. The person in the very front would be the head, and the last one, with a bandana hanging on their clothes, would be the tail. The objective was that the head would have to take the other team’s bandana, being careful that they didn’t lose theirs. The moment the team lost theirs, they would lose and have to stop right where they were.

''How does this relate to the Riboswitch?'' The riboswitch turns the circuit on and off, depending on several conditions. In this game, the team was “On“ while they still had their bandana; they were turned “Off“ when they lost it.

''BASE 3: PROTEIN CODING SEQUENCES'' - This base was one of the hardest to fulfill. We designed a “labyrinth“ of conditions that each team would have to pass through. They would have to dress up with objects in some boxes, and depending on what they wore, they would move through the squares. The one who found the perfect combination would win.

''How does this relate to the protein coding sequence?'' Protein coding sequences, as stated in the Registry of Standard Biological Parts, encode the amino acid sequence of one particular protein. In the game there was only one perfect combination, one combination that would make you win; and you had to find it.

“BASE 4: RBS“ - This activity was somewhat simple, yet fun anyway. We would have two people holding a piece of fabric next to two other people, also holding a piece of fabric, and they would be passing along a small ball, in several different ways, to a small box on the other side of the field. For example, the first time it would be walking and passing the ball however they could. The second time, they would have to throw it as higher as possible, and running. The third time would be running backwards, and the fourth would be jumping on one foot. Only one team got to the fifth time, and they had to do it backwards and jumping on one foot. At the end, the team with the greatest amount of balls inside their box, won.

''How does this relate to the RBS?'' The RBS is the place where the ribosomes bind and start the process of translation. The small balls represent the ribosome trying to get to the RBS, the box.

“BASE 5: REPORTER“ - The activity was quite simple, and at the same time complicated. We had to build 4 boxes with wires, batteries and a light bulb; only two wires would be connected to the batteries and could turn on the light bulb. There were 20 wires, and the first team who found the combination and turn on the light, would win.

''How does this relate to the Reporter?'' The light would turn on whenever the right wires were connected; it acted like a signal. That’s what the reporter does; when the condition is fulfilled, it sends a signal to let you know, just like the light.

“BASE 6: TERMINATOR“ - The last activity was just like a game named Doctor, except that we used balloons instead. Each team would have to get in a closed circle, but instead of holding hands directly, they would hold a large balloon. Then they would have to tangle, without letting go of the balloons, as much as possible. The “doctor“ would then have to undo their “knot“, without breaking the circle. The first ones to blow up or let go of their balloon would lose.

''How does this relate to the terminator?“ The terminator causes transcription to stop; it sort of “breaks it off“. In the game, “transcription“ stopped whenever the person blow up or let go of the balloon.

Conclusion - Return

At the end of the rally, we gave an explanation of the whole circuit; how every part was connected to make it function. We answered questions, we took a picture, and we prized the winner team with a box of cookies. Some of them asked if we had a page on Facebook, and others just thanked us for coming.

One of the girls who was helping us with the rally, was not from the team. She was from second semester, and was really interested in iGEM when she found out about it, so she asked if she could come and help. She was of great support; taking pictures and helping us organize everything. At the end it all worked out, even though the day before we were all going crazy because most of the things were missing. It was a really fun experience.