Overview on result
This page demonstrates the detailed result of the following experimental process.
- Design of the recognition sites
- Testing and filtering the alternative recombinase
- Response curve for the chosen recombinase
- Solution for cytotoxicity
- Design, construction and debug of the three-state ADC
Design of the recognition sites
1. The feasibility of normal open (NO) contact
If we compare our genetic circuit with the electrical circuit, the normal open (NO) in our project is that there is no expression in the normal state and the expression will be activate only when there is recombinase binding on the sites and flips (or cuts) the terminator.
The feasibility of genetic switches based on att-site and unidirectional terminators has been demonstrated in a study published by the Tim Lu’s team in 2013 (see reference). Besides, 2017 iGEM team Peking and Fudan also develop a series of recombinase switches on the same principle.
We replicate the recombinase switch design of 2017 Peking, which is based on recombinase phiC31 with att recognition sites and the unidirectional terminator ECK120034435. We test the expression difference between with and without recombinase. And the result meets our expectation, shown below
Figure 1: the upper shows the mechanism of the NO contact, and the lower
graph shows the feasibility of the NO contact.
2. The comparison between two design of normal close (NC) contact
The normal close (NC) circuit in our project is opposite to the NO circuit, that there will be expression in the normal state and the expression will disappear recombinases bind to the recognition sites. Based on the mechanism of the recombinase, gene translation unit should be placed between two att recognition sites to realize the normally-close control of this gene. To make inserting gene easier, we design BsaI enzyme cutting site between att and the terminator, on the cis-element of our recombinase switches.
At this time, we consider two different design for the recognition sites: placing att sites equidirectionally or placing att sites oppositely, which will cause flipping or cutting respectively. We carry out experiment to test these two designs, the result is shown below.
Figure 2: the mechanism and the result of flipping
Figure 3: the mechanism and the result of cutting
Compare with the cutting design, flipping design has the following advantages and disadvantages:
Advantages:
can proceed the reverse process by using RDF
Disadvantages:the closed (flipped) gene still has the probability of expression
The flipped fragment may have a strong attenuating effect on the expression of downstream gene
After comprehensive analysis, we adopt the design of equidirectionally placing att recoginition sites in most of our following experiment.
Testing and filtering the alternative recombinase
Given the use of more than one recombinase in our system, we increase the diversity of our parts and construct six recombinase-terminator-based switches. We co-transform the G002-based testing plasmids with their corresponding recombinase expression plasmids. After adding IPTG for inducer and culturing in EP tubes for 24 hours, we centrifuge the culturing tubes and make the cells fluorescent under blue light to qualitatively, preliminarily screen our recombinase switches. The results are shown below.
Figure 4: the result of preliminary screening of recombinase system. IBR is the name of our switches (integrase-based relay), followed by the composite parts (attB-TE-attP), integrase – indicates cells without inducing while integrase + means with inducing.
By using flow cytometry, we also screen the recombinase systems in a quantitative way, shown below, whose result is consistent with the above qualitative screening.
The switching performance of normally open contact is quantitatively described by fluorescence attenuation rate of GFP reporter gene in two states. As a reference, we used a plasmid containing only one short unintentional sequence between the promoter and RiboJ as a positive control. The average intensity of bacterial fluorescence was quantified by flow cytometry, and the final value was calculated as follows:
Figure 5: the result of quantitative screening by using flow cytometry
The experimental results of orthogonality are as follows, and the result reveals a fine orthogonality:
Figure 6: The results on the diagonal line, which are yielded from the valid pairs of recombinase and its binding sites, indicate shorter DNA fragments than others in the same row. This proves the fine orthogonality.
Response curve for the chosen recombinase
So far, we have only solved the switching and conduction problems of relay contacts. But as a replay element, it should have a character of changing its ON/OFF state once it reaches rated voltage. We hope our biological relay can have the similar character, which makes it different from the transcription-factor-based, traditional promoter genetic switch.
In the existing applications of recombinase, there is only a simple response to high and low input levels, and the threshold of response is not demarcated. In this study, we mainly explore three issues: whether the recombinase system has a narrow enough hypersensitive response interval to realize state leap (rather than slow change); Determine the factors affecting the hypersensitive response range; Describe the range of different modules quantitatively (that is, parameterized characterization).
We establish a mathematical model based on the thermodynamic equilibrium state hypothesis (see model https://2019.igem.org/Team:GENAS_China/Model ), shown below.
According to the framework of previous studies (see reference), the intensity of Input was used to characterize the concentration of recombinase in cell.
In the following simulation image, the Hill’s coefficient is set to 2 according to the hypothesis of previous studies, and the range of the x axis is determined according to the measurement range of the input strength of the Ptac promoter. The shape of the curve shows that the range of hypersensitivity response is very narrow, which is helpful to realize the response to the slight changes in the input signal.
Different k values will cause the change of the position of the hypersensitive response range in the transfer function of module. The following figures shows how the change in k value affect the transfer function.
Figure 7: different curve under different value of Kp and Kb
In order to make the measurement conditions as close as possible to the theoretical hypothesis, we designed the measurement method 1 based on the steady-state measurement method (that is, recombinase induction first, followed by reporting system induction) and simplified method 2. We use phiC31 recombinase-based relay to compare the two methods and parameterizedly characterize the phiC31 relay system.
Figure 8:
the fitting response curve of phiC31 RBBR component.
The orange part are the parameters that need to be fitted.
Although the fitting results of the data obtained by these two measurement methods are not identical, their transfer functions are very similar. In order to simplify the experimental process, we will adopt the simplified method 2 to conduct the measurement.
Using measurement method 2, we parameterizedly characterize the other two RBBR elements, the parameters are shown below.
Figure 9: the fitting response curve of phiC31, TG1 and INT10 RBBR component.
Solution for cytotoxicity
During the process of experiment, we find that the overexpression of some recombinases will limit the growth of cells. The relationship between gene expression level (represent in input strength below) and the cytotoxicity (represent in OD600 below; the lower OD600, the higher cytotoxicity) is shown in the graph below.
Figure 10: the relationship between OD600 and the input level.
In the figure, INT8 (red) and TG1 (green) have distinct cytotoxicity since the OD600 value drop greatly when the input level increase.
To solve the problem of cytotoxicity, we try to lower the gene expression strength by either changing a weak promoter or finetuning RBS to a weak one. We lower the expression strength in INT8 as an example of changing a weak promoter.
For INT8, when the input is above 30000, the cell growth will be significantly inhibited, and other experiments have proved that the Int8 can lead to sufficient recombination even if the input is the lowest value of this promoter. Suppose our task is to design an Int8 induction system that is not toxic at the highest inducer concentration. To this end, we mutated the original promoter and obtained the following promoter:
Figure 11: different promoter with different relationship between concentration of IPTG and expression of sfGFP. Ptac-M1/M2/M3 are the mutated promoter, which are relatively weaker than the original one.
The combination of promoter ptac-m2 and K3254011 which has the highest output below 20000 is selected, and the results showed that the toxicity was successfully controlled, as shown in the figure below (in blue line), the OD600 value still remains high when the concentration increase (Although the leakage expression is still high, it has been fully recombinated without inducers.)
Figure 12: different result of cytotoxicity before and after the change of promoter.
Therefore, when we evaluate a bio-relay element, we need to evaluate the performance of the whole system (Figure 13)
Figure 13: comprehensive prediction and evaluation of a system’s performance.
Design, construction and debug of the three-state ADC
Based on the biological relay we developed in the project, we built a genetic analog-digital convertor, which can convert the analog input to the digital output. Each recombinase correspondingly controls the on-off of a chromoprotein. Theoretically, the convertor can be infinitely extensive by adding new relays to the system. The performance of the ADC predicted by the model is presented in figure 14 and 15. The amilCP is the first fluorescent protein that is activated by the downstream promoter, and hence it’s constantly expressed in the beginning. amilGFP is then activated during a short period till mCherry start to express.
Figure 14: a genetic circuit demonstration of relay-based ADC
However, the experimental result in figure 15 (upper) doesn’t fit with our simulation. amilCP’s blue color did vanish in expectation but yellow and red color didn’t appear as we predicted. The disappearance of mCherry might be due to the low transcription efficiency caused by redundancy of upstream sequence. But for the disappearance of amilCP, the PCR result showed that at the minimum inducer concentration, amilCP have already been recombinated, indicating that the Int10 might have strong leaked expression in the system. This might due to the upstream phiC31 sequence, an original sequence adapted from phage, which may be multifunctional because of the limited length of DNA in a phage. Hence, there might be one or more potential promoter in phiC31 sequence which may cause the leaked expression of amilGFP
Figure 15: the comparison between stimulation of the performance of ADC (lower) and the experimental result (upper).
Reference
[1] Lou, C., et al., Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology, 2012. 30(11): p. 1137-42.
[2] Zhang, H.M., et al., Measurements of Gene Expression at Steady State Improve the Predictability of Part Assembly. ACS Synthetic Biology, 2016. 5(3): p. 269-273.
[3] Zong, Y., et al., Insulated transcriptional elements enable precise design of genetic circuits. Nat Commun, 2017. 8(1): p. 52.
