Team:ZJU-China/Perspective

1 Most important: Our gene sockets relieve lots of work of scientists.

As shown before, via the methods of lambda-red homologous recombination and new-designed bistable switch, we perfectly achieved our goal to make constructing vectors on chromosomes easier. If we can change the chromosomes as we planned, just through some simple steps, would it sound marvelous?

1.1 The prominent advantages of gene sockets.

Our gene sockets have a lot of prominent advantages and it is superb and unique. The advantages can be divided into three parts, CEO.

1.1.1 Convenience

First of all, as you can see, our gene sockets provide large convenience for adding specific group of genes into specific vectors. It is just like plugging into the power sockets, which is what we do in everyday life, and can be so easy. For example, we have a blueprint in the future, that given a group of genes, maybe 4 or 5, and given metabolic pathways, which decide the logistic relationship among these proteins coded by these genes (even though these genes code promoters and terminators). And our super gene sockets can handle it right away, with little difficulty or effort. What we do is that we just need to standardize every gene, adding standardized ends to them to change them into parts, and put them into cells one by one. Also because of using standardized ends, we try to create a database and software that can help us standardize the given genes into available parts that can be used in our gene sockets (Click here to get GS-BOX!). We’re really glad to invite you to try our software and give us suggestions. What’s more, the workflow of gene sockets will be given on the next section.

1.1.2 Efficiency

Secondly, our gene sockets have high accuracy and high efficiency for constructing a vector. The high speed depends on the high efficiency of the homologous recombination we’ve chosen. Also, the high accuracy results from high accuracy of this homologous recombination. We chose lambda-red dependent homologous recombination as our tools to build gene sockets, and it is one of the most efficient and mature ways to insert genes in prokaryotes. As we came up with this idea, we thought about the process that we could inset one gene by one day. Maybe, as the efficiency of lambda red could be high, we can insert genes faster than we expected. It can totally improve the efficiency and save time of scientific researches in the future.

1.1.3 Originality

Thirdly, we have an exquisite and original design without resistance screening. We add green and red light (sounds like traffic light) into this system to detect and verify whether the given gene has inserted into the specific spot. In this part, we use a new but great bistable switch to achieve this signal system. Our novel bistable switch is totally different from the traditional bistable switch we’ve seen for many times. It’s more stable and accurate. Through this signal system, do it step by step, we can confirm that every gene has been integrated successfully through every change between red and green light. However, that doesn’t mean that we can just use the fluorescence system. Our two reporters can be various, such as resistance (When the bacterium of interest count a small part of the whole bacterium, resistance can be a good screening way.), blue-white plaque selection and so on. We can choose the adequate reporter with our research. Also, compared to the traditional restriction enzyme digestion and ligation ways, our gene sockets provide a chance to make a no gap linkage and sometimes it helps a lot.

What’s more, compared to the traditional lambda-red homologous recombination dependent gene knockout method, our novel homologous recombination based GeneSocket can be said that it excels its predecessor.

Period	Traditional lambda-red gene knockout method	GeneSocket
Preparation	Gene of interest are going to be inserted.
	Insertion site should be found, a series of plasmids are used to acquire resistance gene with FRT or LoxP site.	All design and preparation steps are designed by GS-BOX.
Recombination	Insertion mode: Gene is linked to resistance gene, and two recombination sites are flanked on each side.	With standard helper parts BBa_K1433013, no resistance gene shall be used in recombination. Helper parts are also shorter than resistance gene to spare more space for GOI.
	Plasmid like pKD46 (heat sensitive) carry Lambda red gene to allow recombination happened.	In GeneSocket, and Support device carry Lambda red gene.
Resistance gene retrieve	pKD46 should be discarded after this step, and a new plasmid like pCP20 carrying recombinase is transformed into cell to retrieve resistance gene.	Since no resistance gene are used in insertion fragment, there is no need to consider gene retrieving. All things can be done in one plasmid no repeatedly selection shall be done!
	Scar can’t be avoid via this method of the recombinase site.	Scar can be avoided if necessary, by careful design of the recombination site.

1.2 The workflow of using gene sockets. (Fantastic guide)

1.3 The product we can provide with.

2 Can be used in different species.

Our gene sockets idea provides a wide application in all kinds of creatures. Except for prokaryotic cells, it can also outreached to eukaryotic cells. We’ve learned another ways of homologous recombination depending on CRISPR, and it is really possible to build gene sockets in eukaryotic cells instead of lambda red recombination.

2.1 Prokaryotic cells

As shown before, our gene sockets can be perfectly used in prokaryotic cells via lambda-red homologous recombination and tyrosine recombinase bistable switch methods. The workflow and software are available in the former section.

2.2 Eukaryotic cells