Overview

Members of Peking iGEM 2017 team devoted themselves to constructing a part library related to recombinases. We have not only submitted 5 basic parts consisting of improved recombinases and recombinase-RDF fusion proteins, but also provided 31 composite parts including a series of unidirectional terminators flanked by attB/P sites and some well-performed constructs to express or report recombinases.

Functions of all the parts were tested and validated both in controlled laboratory condition and in normal condition. They can be used as powerful tools for other iGEM teams.idirectional terminators flanked by recombination sites.

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Favorite Basic Part

Introduction

This year, our team has selected a series of recombinases through literature searches and characterized a number of them. Here we present the best characterized recombinase - Bxb1 gp35 (BBa_K2243012) as our best basic part for the award.

With well-behaved inversion performance, Bxb1 gp35 can invert the target DNA sequence efficiently, thus changing the function of the circuit according to our demands, and can be designed and used by other iGEM teams in their project/research.

Figure 1. Schematic of the standard approach used for recombinase characterization

How we characterized and improved the part

Using our standard approach to characterize recombinases (Figure 1), we selected two different vectors and a series of RBS to express Bxb1 to invert the constitutive promoter BBa_J23119 and change the fluorescence. Through the screening procedure, we improved the performance of Bxb1 gp35 and obtained a number of well-behaved matches, among which Bxb1 gp35 recombinase works particularly well. (See more in Flip-flop)

Properties

Through the fine tuning, we improved the performance of Bxb1 gp35 efficiently. Here we present some of the well-tested behaviors of this recom-binase, which indicates the excellent properties of Bxb1 gp35.

High efficiency of inversion under most conditions

Whether with computer-designed RBS or widely used RBS from the iGEM registry, Bxb1 exhibited a relatively high efficiency in inverting the target promoter.

Low leakage through tuning

Through our tuning procedure and exhaustive characterization, we ob-tained a series of response curves to the concentration of inducer, in which relatively low leakage (no inducer) and high inversion efficiency (proper in-ducer concentration) were found. This property meets our requirements well, indicating that Bxb1 gp35 can change the DNA accurately according to our needs.

Potential for controlling the downstream circuit

In addition to inverting the promoter to change the gene expression, we also used the improved Bxb1 expression plasmid to invert a unidirectional terminator (See more in controller), thus opening or closing the expression of a downstream reporter gene.

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Best Composite Part

Our team constructed many composite parts for the expression of the reporter and the recombinase. Here we present a list of them, and introduce the best-performing part: Bxb1 attB_ ECK120034435F_Bxb1 attP , as our best composite part.(See more in controller)

Function of Bxb1 attB_ECK120034435_Bxb1 attP

Figure 1. Schematic of the Bxb1 attB_ECK120034435_Bxb1 attP composite part as integrated into the GFP expression plasmid.

Our best composite part consists of the Bxb1 gp35's target sites attB/attP and one well-behaved unidirectional terminator (ECK120034435) between them. This construct can be regarded as an outstanding biological switch for RNA polymerase, because of its high ratio between forward and reverse terminator strength. What's more, we can express Bxb1 gp35 to change the direction of the terminator and switch on/off the expression of downstream genes.

Excellent performance

High ratio between forward and reverse terminator strength. (Over 1000-fold)

The terminator ECK120034435 is the best-performing unidirectional terminator in our library, and its Ts ratio is higher than 1000-fold, which indicates that it can potentially be used as an RNAP switch in our circuit.

No latent reactivity to transcription.

Unlike some other terminators we observed and characterized, ECK120034435 has no latent promoter reactivity under most conditions, and won't start transcription even if ligated with recombinase reaction sites. Thus, this terminator can be used as our controller to improve the predictability of our constructed circuit.

Robustness under different conditions.

When we introduced one or two pairs of attB/P sites on both sides of ECK120034435 to construct the controller that can be inverted by recombinase, the great performance of this unidirectional terminator was robust. This property shows its potential utility in constructing more complex control circuits in our sequential logic circuitry.

Ability to control expression through the recombinase.

With the excellent performance in opening/closing the transcription process, we have proved that our best composite part can be inverted by Bxb1 gp35 to obviously change the GFP expression, which shows that this construct is potentially a great control unit for gene expression under the control of recombinases.

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Part Collection

Members of Peking iGEM 2017 team devoted themselves to constructing a part library of unidirectional terminators flanked by recombination sites.

This construct can be regarded as one outstanding biological switch to RNA polymerase because of its high ratio between forward and reverse Terminator Strength (Ts). What's more, we can express the corresponding recombinase to change the direction of terminator and switch on/off the gene expression downstream.

The collection of parts mostly has good performance and can be improved further. Their Ts ratios are relatively high (some even higher than 1000 folds), which indicates that they can be used as great RNAP switches in our circuit potentially. Some of them have robustness even when ligated with attB/P sites and we have observed obvious inversion through the expression of recombinase which indicates the usability to control the expression through recombinase.

Our part collection includes:

Bxb1 attB_Unidirectional terminator_Bxb1 attP

Bxb1 attB_L3S3P22F_Bxb1 attP(BBa_K2243018)
Bxb1 attB_L3S3P22R_Bxb1 attP(BBa_K2243019)
Bxb1 attB_ECK120030221F_Bxb1 attP(BBa_K2243020)
Bxb1 attB_ECK120030221R_Bxb1 attP(BBa_K2243021)
Bxb1 attB_ECK120033737R_Bxb1 attP(BBa_K2243022)
Bxb1 attB_ECK120034435F_Bxb1 attP(BBa_K2243023)
Bxb1 attB_ECK120034435R_Bxb1 attP(BBa_K2243024)
Bxb1 attB_ECK120010855F_Bxb1 attP(BBa_K2243025)
Bxb1 attB_ECK120010836F_Bxb1 attP(BBa_K2243026)
Bxb1 attB_ECK120010836R_Bxb1 attP(BBa_K2243027)

phiC31 attB_Unidirectional terminator_phiC31 attP

phiC31 attB_ECK120034435F_phiC31 attP(BBa_K2243031)
phiC31 attB_ECK120034435R_phiC31 attP(BBa_K2243032)

TP901-1 attB_Unidirectional terminator_TP901-1 attP

TP901-1 attB_ECK120030221F_TP901-1 attP(BBa_K2243033)
TP901-1 attB_ECK120030221R_TP901-1 attP(BBa_K2243034)
TP901-1 attB_ECK120034435R_TP901-1 attP(BBa_K2243035)

int2 attB_Unidirectional terminator_int2 attP

int2 attB_ECK120030221R_int2 attP(BBa_K2243028)
int2 attB_ECK120010855F_int2 attP(BBa_K2243029)
int2 attB_ECK120010855R_int2 attP(BBa_K2243030)

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Improvement

Though there are already many recombinase parts in the iGEM registry, quantitative characterization data on them is very limited, and what's more, there is no single standard characterization method for serine recombinases. In addition, even though the attL/R sites formed by the recom-binase inversion are not recognized by the recombinases which create them, we successfully achieved the "reset" process using fusions of the recombinases and their recombination directionality factors (RDF).

Improvement of TP901-1

The submitted TP901-1 integrase part(BBa_K1132010) in the iGEM registry only consists of a brief introduction to it. The only data for TP901-1 was obtained through the dimerization of split integrase, which did not work so well. Additionally, there appears to be no quantitative information to evaluate the RDF of TP901-1.
This year we submitted the TP901-1 (BBa_K2243000) coding region as a basic part, and obtained detailed data to evaluate the inversion efficiency of TP901-1 integrase with different RBSs and vectors through our standard characterization method. This opens the possibility to tune and improve its performance further to meet our design requirements.
We also used our composite part BBa_K2243035, comprising the unidirec-tional terminator ECK120034435 flanked by attB/P sites, as the reporter to evaluate the performance of TP901-1, which broadens the prospects to use TP901-1 to control gene expression.
We also constructed the fusion protein BBa_K2243014, comprising TP901-1 integrase and its RDF(BBa_K1733000), and tested its ability to invert DNA through attL/R sites. (See more in Flip-flop)

Improvement of Bxb1 gp35

Some characterization data of Bxb1 gp35 (BBa_K907000) is already available in the iGEM registry, but it is still limited because the RBS plays a significant role in recombinase expression and performance according to our modelling and observations. Without proper RBSs, Bxb1 may exhibit very high leaky expression (even approaching the induced expression level).
This year, our team constructed an RBS library to improve the efficiency of Bxb1 gp35(BBa_K2243012). We obtained a number of well-behaved matches (from BBa_K2243002-BBa_K2243005), and measured their corresponding transfer curves against the concentration of inducer. Through our improvements, Bxb1 showed a very high efficiency of nearly 95% and low leakage of inversion.
Additionally, we used our composite parts (BBa_K2243023 and BBa_K2243024), comprising the unidirectional terminator ECK120034435 flanked by attB/P sites as the reporter to evaluate the performance of Bxb1 gp35, which broadens the prospects to use Bxb1 to control gene expres-sion.
Finally, we fused Bxb1 gp47 (BBa_K907001) with Bxb1 gp35 and tested the ability of this fusion protein (BBa_K2243013) to invert the DNA sequence flanked by its specific attL/R sites.

Improvement of phi31 Integrase

Though there are quite a few parts related to phiC31 integrase, there is no systematic standard characterization of it. The only relevant information we found comprises fluorescent images that show the performance of phiC31 in plants, which is not quantitative and accurate.
Our team used our standard reporter (BBa_K2243008) to characterize phiC31, and selected a few well-behaved phiC31 translational units which can achieve very high inversion efficiencies with low leakage. Through these data, we will be able to make use of phiC31 in our future designs more efficiently and accurately. Additionally, we used our composite part (BBa_K2243031), consisting of the unidirectional terminator ECK120034435 flanked by attB/P sites, as the reporter to evaluate the per-formance of Bxb1 gp35, which broadens the prospects to use Bxb1 to con-trol gene expression.

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Team:Peking/Parts