Part:BBa_K3445002
RFP included with Vibrio Compatible Plasmid Backbone
RFP attached to the mutated vibrio compatible plasmid backbone (BBa_K3445000) in order to decrease replication time.
Successful Replication of reverted pSB1C3 in Vibrio Natriegens (Alma 2020)
We have been unable to transform J04450 into Vibrio natriegens, despite being able to get other ColE1 and pUC19 origin based plasmids in this species. We have discovered that pSB1C3 contains a point mutation in the origin of replication that could explain this effect. This part is a reversion of this mutation, making pSB1C3 into a true pUC19 origin plasmid. We proceeded to test this by transforming by electroporation into Vibrio natriegens, as well as measuring plasmid copy number.
The results above are representative of several experiments. As can be seen, J04450 cannot be transformed into Vibrio, while our mutant was able to successfully produce colonies. In these results, “-“ is the negative control plate streaked with pSB1C3 containing J04450 (“RFP”). This is the most commonly used backbone for iGEM BioBricks. The plate contains no growth. “+” contains pGGA without the plasmid that expresses RFP. This contains the pUC19 origin, which has expression in vibrio. However, pGGA is not compatible with iGEM BioBricks. “Mr” (BBa_K3445002) is the mutated backbone (BBa_K3445000) to both contain the gene needed to express RFP and allow for compatibility to show expression in vibrio. We observed in these experiments a red color in Vibrio, indicating that the bacteria can express the RFP gene.
We also measured the plasmid copy number of our mutant, both in E. coli and in Vibrio. We did so by performing qPCR on DNA isolated from equal numbers of these bacteria using either genome targeting primers (dnaE) or plasmid targeting primers (oriQ), as done in previous studies. The copy number is thus measured as 2 raised to the difference between the oriQ Ct value and the dnaE Ct value.
In E. Coli, the copies of plasmid per genome for J04450 with a pSB1C3 backbone was 6.92 copies as measured by qPCR. For the mutated backbone containing J04450 (“K3445002”), the plasmid per genome measured was 12.21, 25.11, and 10.85 copies. This averages to roughly 16.05 copies of plasmid per genome shown in e. Coli for K3445002. In vibrio, the reported values for plasmid per genome for pGGA was found to be 306 copies. The found values for copies of plasmid per genome for K3445002 in vibrio could not be determined in practice due to there not being a reported Ct value for K3445002 with the DNA E primer. However, an estimated value was determined by using the Ct value for pGGA with the DNA E primer and the Ct value for K3445002 with the OriQ primer. The estimated value for K3445002 in vibrio was determined to be 6.73 copies of plasmid per genome. These values indicate that K3445002 is effective in both E. Coli and Vibrio natriegens, and there may even be a higher plasmid copy number in E. Coli with K3445002 in relation to the copy number for J04450. The dissociation curve was analyzed to verify that each reaction in the qPCR only produced one product. Taken together, these results show that we have improved the backbone of all parts (and J04450 specifically), resulting in a plasmid that is still capable of replication in E. coli, but now also available for use in Vibrio natriegens. Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 2049
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