Team:DTU-Denmark/Design Plasmid
Vector Design
Creating a promoter test platform for A. niger
As there were no suitable devices in the iGEM registry for testing promoters in filamentous fungi, our team had to develop our own test platform for easy cloning and characterization of promoters. Our solution was simple: We built a new reporter device by placing an exchangeable placeholder promoter upstream of an ORF expressing a fluorescent protein. We elected to use mCherry as our reporter protein because it is visible to the naked eye in E. coli and chemical standards exist, which will ease comparisons between plate readers and labs. Our choice of mCherry is described in greater detail on the Measurement design page.
Using the new iGEM Type IIs RFC[1000] standard, we ensured that a placeholder promoter would be able to be cut out from the test device using BsaI, which would leave fusion sequences on the test device strands that would be compatible with level 0 MoClo promoter+5’UTR parts.
The whole test device is flanked by two of the Unique Nucleotide Sequences (UNSs), originally identified by Torella et. al. 2014 [2]. As the name suggests, these nucleotide sequences have been selected on the basis of being unique, i.e. these sequences do not have a direct match anywhere in the E.coli genome. When using E.coli as a host organism for constructing synthetic circuits, these UNSs can therefore be used for Gibson cloning or other kinds of homology-based cloning methods. Alternatively, the test device can be flanked by SapI sites, making the whole transcriptional unit of the test device be able to be integrated into any even plasmid when using the loop system. [1]
Self-replicating plasmid
The widely-used promoter+RBS part BBa_R0010 was used as the placeholder promoter in our design since this prokaryotic promoter would be able to express our mCherry reporter in E.coli, but be unable to express mCherry in our fungal host. This will make it easier to screen for correct insertion of eukaryotic promoters into the test device, as transformants with the correct insertion of promoters will appear white on transformation plates, whereas an unsuccessful replacement of the placeholder promoter will yield red colonies. This was done to decrease the time used for screening transformants using tissue PCR by implementing this first screening method.
This test device was used to evaluate all the promoters designed during this project.
To obtain expression from the test device in A. niger, an initial plan was made to integrate the test device into an already existing plasmid backbone, pDIV078, which was provided by Zofia Dorota Jarczynska from DTU Bioengineering. The pDIV078 plasmid is able to replicate autonomously due to the presence of an AMA1[3] sequence (short for Autonomously Maintained in Aspergillus). Besides the AMA1 sequence, the pDIV078 plasmid contains an ampicillin resistance gene for easy selection and propagation in E. coli.
Self-replicating plasmids have some advantages compared to genomic integrations, such as a faster transformation, lower concentrations of DNA are required, and the expression is not dependent on chromosomal context and histone patterns. However, there are also some disadvantages to using plasmids instead of genomic integration. Two major advantages of using genomic integration in the characterization of promoters is that the inserted DNA cannot easily be lost if the selective pressure is removed and that the copy number is tightly controlled at one, a fact that many experts we talked to also pointed out.
Genome integration of promoters.
As described above, genome integration of the promoters tested in this project is advantageous for several reasons.
The second design iteration of our promoter test system therefore included a new plasmid design that would allow the test device to be integrated into the genome of A. niger.
As our first proof-of-concept that it was possible for us to accomplish genome integration and subsequently screen for correct transformation, we designed an integration vector named pGIA2P_1 (plasmid for Genome Integration in Aspergillus at integration site 2, using PyrG as the selection marker). The pGIA2P_1 plasmid consists of the same pyrG gene used for our AMA plasmid design, flanked by two integration site sequences as described by Holm, D. K. (2013) [4]. This specific integration site targets the albA conidial pigment gene of A. niger, and insertion of our test device into the IS2 site will therefore disrupt the ability of the transformants to produce black pigment, making the conidia appear white rather than black for successful transformants. This allows for an early selection of successful genomic insertions without resorting to performing tissue PCR or some other screening method.
After the first proof-of-concept, a more advanced version of the integration plasmid was constructed. This new plasmid, named pPEA2P_1 (For Promoter Evaluation Aspergillus integration site 2, PyrG selection, variant 1), was designed to test if it was possible to both integrate and express a fluorescent reporter in A. niger.
Future Design
We have already made designs for future improvements of our promoter test system. First of all, we have made plans to domesticate several of the test plasmids used in this project. Furthermore, we would like to expand the genomic integration plasmids to allow for the easy exchange of promoters, just as the self-replicating plasmid we have developed. This will allow us to characterize the promoters with even greater accuracy and precision.
We would also like to expand on our design by introducing a second fluorescent reporter protein, most likely moxGFP as described here which is controlled by a well-characterized promoter. This internal standard will allow us to benchmark all promoters against the well-characterized promoter and thus eliminate even more variables during characterization.
Lastly, we have designed all our parts to not contain any SwaI sites. SwaI is a blunt-end restriction enzyme, that can be used to linearize the promoter test circuit without using PCR. This removes the risk of introducing mutations during the PCR and it also serves as a more cost-effective way of linearizing the construct.
(1) B. Pollak et al., “Loop assembly: a simple and open system for recursive fabrication of DNA circuits,” New Phytol., vol. 222, no. 1, pp. 628–640, 2019.
(2) J. P. Torella, C. R. Boehm, F. Lienert, J. H. Chen, J. C. Way, and P. A. Silver, “Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly,” Nucleic Acids Res., vol. 42, no. 1, pp. 681–689, 2014.
(3) D. Gems, I. L. Johnstone, and A. J. Clutterbuck, “An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency,” Gene, vol. 98, no. 1, pp. 61–67, 1991.
(4) M. T. Nielsen et al., “Heterologous Reconstitution of the Intact Geodin Gene Cluster in Aspergillus nidulans through a Simple and Versatile PCR Based Approach,” PLoS One, vol. 8, no. 8, 2013.
