The exploration of application of a yeast toolkit for Multi-part Assembly
Just as what we demonstrated in previous part, our project aims to construct two complete plasmids to initiate the metabolism pathway to convert L-tyrosine into catechin. This process involve assembly of nine coding sequences and ten more promoter and terminator sequences, which results in a complicated multi-part assembly. To overcome this obstacle, what we need is a set of toolkits for a convenient multi-part Assembly.
After some research, we found a highly characterized Yeast Toolkit for modular and multi-part assembly. The related article is published by Lee M E et al. In general, the toolkit provides a series of sequences, including promoters, terminators, connectors, selective markers in either E. coli and Saccharomyces cerevisiae and other functional sequences. By using this toolkit, we can operate standardized and flexible plasmid construction through Golden gate construction.
Fig.1Yeast toolkit starter set of 96 parts and vectors
What we do to this toolkit is that we test the performance of most of parts in this toolkit by analyzing the yield of catechin and naringenin. Besides, we also explored the optimum temperature for assembly program of each individual parts in yeast toolkit. This can be seen in our Protocol page. What’s more, we also try to add an extra terminator, tCYC1 (BBa_K4012023), to enrich the variety of the terminators in it.
In a nutshell, all we have done has paved the way for other teams who want to use S. cerevisiae to carry out multi-parts assembly, verifying the reliability and availability of this toolkit. Our results do benefit other team, for example, we introduced this toolkit to team Links_China and they managed to achieve their engineering goal using this toolkit. More details view:
The design and assembly of the naringenin sensors
During the experiment, the products of our first half pathway, naringenin, require HPLC tests to measure the concentration in different situations of naringenin production. The HPLC experiments is fairly time-consuming and complicated. Therefore, we want to find a way for faster initial detection of naringenin.
What inspire us is the research done by Siedler S et al in 2013. The article takes FdeR sequence to express FdeR protein dimer. In the presence of naringenin, FdeR probably binds to a box located upstream of the FdeR promoter and activates cfp expression. (Fig.2)
Fig.2 The basic mechanism for the sensor
Meanwhile, we find a composite part, BBa_k1497020, constructed by iGEM14_TU_Darmstadt. However, when we view the sequence of their sensor, we found that they put FdeR sequence and sfGFP sequence in same direction. In this occasion, the cfp gene will perform either there is naringenin activating the inactive protein performed by fdeR or not, neutralizing the sensor. Based on this finding, we design the sensor again with FdeR and sfGFP sequence in opposite direction followed by the article. In this way, we can obtain a sensor with practical use. (Fig.3A&3B)
Fig.3 A: Incorrect sensor design; Fig.3 B: New design of the sensor, pay attention to the direction of the sfGFP sequence.
We managed to assembly the new sensor in complete plasmids, followed by sequence analysis. Despite the fact that the limitation of time does not allow us to do further test on the ability of the sensor in detecting naringenin, we still verify the feasibility of assembling such a sensor, which provide valuable results for following teams that want to apply the sensor or construct similar composite part.
Fig.4 A: The result of sequence analysis of assembly of original sensor plasmids; B: The result of sequence analysis of assembly of improved sensor plasmids
 Lee M E, Deloache W C, Cervantes B , et al. A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly[J]. Acs Synthetic Biology, 2015, 4(9):975-986.
 Siedler S, Stahlhut S G, Malla S, et al. Novel biosensors based on flavonoid-responsive transcriptional regulators introduced into Escherichia coli[J]. Metabolic Engineering, 2014, 21:2-8.