Team:ZJU-China/Design

1.BBa_K1433001 attB-BBa_J23110-attP

BBa_J23110 is a middle strength promoter from the constitutive promoter family, which are flanked by attB and attP sites, constructing the core parts of bistable switch. The direction of the promoter, which is controlled by Int and Xis, decides the state of support device is controlled (See bistable switch page for detailed information.)

2. BBa_K1433005 Lambda red CDS

Lambda red CDS from pKD46. See this page for its composition and function.

3. Bireporter expression device

Reporter 1 & reporter 2 are paired to vividly reflect the direction of promoter so as to select specific cell. Generally, there are two states in this bi-reporter expression device:

State 1: WORK State

Lambda red & reporter 1 is expressed, and support device is turned on to recombine parts.

State 2: SELECT State

Only reporter 2 is expressed, which is used to select cells that have inserted parts.

There are two kinds of combination of reporters: fluorescent proteins or antibiotic resistance genes. For the former, it is easy to get results through fluorescent tests or even by naked eyes. Besides, cells can also be measured or counted accurately by using flow cytometry, which we used in testing our bistable switch and state stability. For recombination selection, resistance gene is more reliable actually because it selects candidate cells through a survival/death mode. Even the successful recombination cells are at a very low concentraion after electrotransformation, they can stilled be selected and assembled after plate cultivate.

4. BBa_I0500 + BBa_1433003

Xis is controlled by pBAD promoter. When arabinose is added, Xis will be expressed, while only if Int exists will the support device turn to reporter 1 and the Lambda red system restarts.

AAK tag is added after Xis gene, which can accelerate the degradation rate of Xis, avoiding the interference of Int expression on the next round of attB/attP turn^[1].

5. Plasmid backbone

The previous pKD46 plasmid uses a pretty good initiator oriR101 & repA101-ts (BBa_K524000) which is a low copy origin (5-10) of replication derived from the pSC101 replication origin. It is also a temperature-sensitive initiator which loses activity significantly at 37°C, totally inactivated at 42℃. Therefore, with this initiator we can easily discard the support device when circuit construction is over.

Low copy number of the support device also allows the concordance of bistable switch. Since a single plasmid carries a single switch, it is easy to find that the more plasmids there are, the harder all these switches can keep in the same state. There is no need to be afraid of that the low copy number may eliminate the expression of Lambda red gene or Xis protein since we can make up by using stronger promoter and RBS, These proteins are not needed in high amount, and over expressing some of them will even lead to inhibition of cell growth.

1. Promoter BBa_J23110

The promoter we use here has two functions:

1. To promote transcription of Int after bi-terminator unit is replaced by the inserted circuit.
2. To promote the inserted circuit CDS if needed.

BBa_J23110 is a middle strength promoter from a constitutive promoter family. It can be replaced if users do not need it, but users should notice that there is a promoter behind bi-terminator unit which allows Int expression. (See solutions below)

2. Bi-terminator unit

This is the basic unit of the site of GeneSocket.

The orange square here represents the Lambda red recombination site. Bi-terminator (BBa_B0015) is able to firmly terminate Int transcription. When the inserted part replaces the terminator, no termination exists and Int gene will be expressed. rep-Int-exp.gif

3. Int expression unit

Recombinase Int can flip over attB/attP sites ( see bistable switch page). Besides, if Xis protein exists, Int + Xis can flip over attL/attR sites. It is crucial to tune the expression of Int to a suitable level that can reach the following balance:

1. Enough to flip over all attB/attP sites on the support device.
2. If Int cannot be over expressed otherwise there may be not enough Xis to combine with all the Int to flip over attL/attR site, causing Int+Xis and Xis function respectively, which will lead to a “Stoichiometry mismatch” (Low Xis to Int ratio can lead to bidirectional DNA inversion.)[1]

4. The location of socket:

Our socket is inserted into bacterial chromosome in advance before any circuit construction. Therefore, at the very beginning, the socket itself is like an “insertion parts”. We use pKD46 to recombine our socket into a safe site on LacZ gene of the strain Escherichia Coli DH10B. Since LacZ is an unnecessary gene, the loss of LacZ will not have any influences on the growth of cells. Still, different locations on chromosome could have different environments ^[2], which further research can be down to improve.

1. Homology region of inserted part.

The homology regions flanking beside inserted parts are added by PCR. The Latter parts must have SpeI restriction site to link with our standard parts.

2. Restriction sites

The inserted part is cut by SpeI after PCR and standard parts are cut by XbaI and PstI. After ligation, no restriction site exists between the inserted part and the standard part. *SepI and XbaI are isocaudomer.

3. Bi-terminator regions

The bi-terminator region is inverted in standard parts. The terminator we choose here is a unidirectional terminator. It only works after Int and Xis flipping over the attL/attR site.

4. The last homology region

The last homology region is a special site which will not change during circuit construction.

5. Additional promoter

If the inserted part has terminator, an additional promoter should be added behind it to allow Int expression. This promoter here is BBa_J23110. You can also change the promoter or use you own promoter. See Solution page for more information.