Part:BBa_K4849019:Design
PrnpB->eYFP expression construct for Synechocystis sp.
Design and Experimental confirmation
Our intention was to use the PrnpB-eYFP-TrrnB construct as a reference, when characterizing the PcopM195-BCD zinc-inducible Synechocystis promoter by measuring eYFP fluorescence in transformed Synechocystis sp. PCC 6803 cells. We performed the Level T assembly using BbsI enzyme (Vasudevan et al., 2019; Gale et al., 2019) to ligate L1P1 construct containing the PrnpB-eYFP-TrrnB transcriptional unit (BBa_K4849007), and the Level M plasmid containing the end-linker 1 (BBa_K4849017) required to provide the 3’ overhang for assembly into a Level T pSEVA421-T replicative vector (https://www.addgene.org/119555/) for expression in Synechocystis. The assembled Level T PrnpB-eYFP-TrrnB construct was transformed into competent Escherichia coli TOP10 cells. Colony PCR was used to screen for colonies with the correct insert size. The expected amplicon size was 1494 bp. Four of five colonies gave the expected size band with colonies D and E giving the strongest bands (Figure 1).
Figure 1. Colony PCR of Level T eYFP constructs.
Two colonies (D and E) with the correct cPCR band were screened by restriction digestion with BciVI (cuts out the insert) and AvaI (linearizes the plasmid), and the digested DNA was analysed by gel electrophoresis. After restriction digestion, while the correct number of bands was observed, they were all larger than expected (Figure 2). Due to time constraints, we decided to continue with Synechocystis transformations.
Figure 2. Restriction digestion of Level T eYFP constructs.
We also sent the Level T PrnpB-eYFP-TrrnB construct to Edinburgh Genome Foundry for sequencing by Oxford Nanopore technology and analysis. The sequencing analysis report showed that most reads of the Level T PrnpB-eYFP-TrrnB construct (barcode34/ lvT_Prnp_eYFP) were of the approx. expected size. The ‘coverage plot’ showed that the entire plasmid was observed, and no large insertions were detected, which confirmed that the construct was correct despite larger than expected size of bands observed after restriction digestion, suggesting it was an experimental error. From comparison with reference sequence (Figure 3), some ‘True’ mutations were detected in the backbone of the construct, and there was a potential double C>T mutation at locus 543 of the eYFP CDS, however, due to low coverage of this part of the sequence this is not conclusive.
Figure 3. The table lists mutations detected in the Level T PrnpB-eYFP-TrrnB construct. Those highlighted in red are ‘True’ mutations, whereas those not highlighted are sequences known to be challenging to accurately sequence with nanopores (usually secondary structures / repetitive sequences). In this table, DP is how many reads cover this part of the sequence, RO is how many of those reads contain the expected sequence, AO is how many contain an alternate allele.
We performed the Synechocystis transformation protocol received from ABOA-Turku’s iGEM team with some modifications. First, the main Synechocystis sp. PCC 6803 culture used for transformations was a mixture of two cultures that were at OD630=0.8, and not at OD750=0.8 (mid logarithmic growth phase) as suggested in the original protocol. Second, the recommendation was to keep plates at 30°C and 50 µE until slight green film arose (ca. 2-4 days), however, we only had a shaking incubator with a light source at our disposal, so we kept the plates there. Synechocystis transformations failed - there was no green bacterial film on the plates after a week, the plates started drying out and had to be disposed of.
References
Gale, G.A., Osorio, A.A.S., Puzorjov, A., Wang, B. and McCormick, A.J., 2019. Genetic modification of cyanobacteria by conjugation using the CyanoGate modular cloning toolkit. JoVE (Journal of Visualized Experiments), (152), p.e60451.
Vasudevan, R., Gale, G.A., Schiavon, A.A., Puzorjov, A., Malin, J., Gillespie, M.D., Vavitsas, K., Zulkower, V., Wang, B., Howe, C.J. and Lea-Smith, D.J., 2019. CyanoGate: a modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax. Plant Physiology, 180(1), pp.39-55.
Source
PrnpB originally from Synechocystis sp. but we synthesised; eYFP-synthetic; TrrnB-synthetic;End-linker - synthetic
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1101
Illegal SapI site found at 4