Goals

Overview of plasmid design

In our cloning experiments we worked with two different plasmid backbones, pJUMP26-1A and pSB1C30. pJUMP26-1A is a plasmid used in bacterial expression and synthetic biology with a medium copy number, one origin of replication, p15A, and resistance to Kanamycin. pSB1C30 is a high copy plasmid with one origin of replication, pMB1, and resistance to Chloramphenicol. The representation below schematically illustrates each step of the biobrick construction process.

Figure 1: insertion of promoter palkB into pSB1C30 (left) and insertion of J23119 into pJUMP26-1A (right)

Figure 2: insertion of alkB1 into pSB1C30-palkB (left) and insertion of alkS into pJUMP26-1A-J23119 (right)

Figure 3: insertion of the J23119+alkS construct into pSB1C30-palkB-alkB1

The final construct is introduced in pSB1C30:

Figure 4: pSB1C30 with inserted fragments-palkB+alkB1, J23119+alkS

How it works

In order to obtain a plasmid that would have the petroleum degradation property, we designed a system with two genes and two promoters. AlkB1 can, when exposed to NADH-rubredoxin reductase and rubredoxin, catalyze the hydroxylation of n-alkanes and fatty acids. This gene corresponds with the published sequence from Alcanivorax borkumensis. AlkS is an alkane-responsive transcriptional activator. As it is located upstream of alkB1, it activates the expression of alkB1. However, this has not yet been tested for A. borkumensis. Although the alkS transcription factor is described and it’s known to induce alkB1 expression, the sequence of the promoter region is not described. Therefore, we had to look for a transcription factor and a promoter from a different organism. For the organism Pseudomonas putida the alkS gene has the same activation mechanism as for alkS in A. borkumensis but it’s much better documented. The alkS sequence expressed in A. borkumensis shows a 48% amino acid identification with the one expressed in P. putida. This is a visual representation of the alkane degradation mechanism of alkB1 and alkS:

Figure 5: Alkanivorax borkumensis petroleum response. Created with BioRender.com

Promoters

We considered multiple options when choosing our promoters, but in the end we decided for the ones below based on literature reviews. J23119 is an artificial BioBrick promoter found in the iGEM registry of Standard Biological Parts. It’s part of the J23 series, but the J23119 was found to be the strongest and it has been used in several cyanobacteria, including PCC 7942, one of the strains used in our experiments. It was discovered that from several promoters tested, J23119 resulted in the highest enhanced yellow fluorescent protein expression levels [1]. PalkB is an alkS-inducible promoter for alkB1. The sequence that we ordered corresponds with nucleotides 7510-7368 from P. putida. Expression of this promoter is regulated by the carbon source used, however this is different for alkS as this gene is transcribed similarly for multiple sources of carbon. This way, the expression of the promoter can be modulated by variable transcription levels of alkS [2].

The Plasmid Backbone

When we first started drafting a plan for our cloning experiment, we were thinking of inserting the alkB1 gene and the alkS-inducible promoter into the pJUMP28-1A backbone and the alkS gene and the J23119 promoter into the pSB1C30 backbone, resulting in two different plasmids that could later be introduced into our targeted organisms. However, due to the fact that both plasmids had the same origin, pMB1, they co-exist in the same cell. Not to mention that high-copy plasmids, like the ones we had picked, contained origins of replication from family A, which are only compatible with families B and C. But the origins of replication from families B and C are low copy. This means that the expression of the proteins inside of the plasmids will be directly proportional to the number of plasmids inside the bacteria. Alternatively, palkB promoter and alkB1 should be inserted into pSB1C30, and the J23119 promoter and alkS into a different plasmid, pJUMP26-1A, therefore having two plasmids with different origins of replication.

References

[1] Huang C-H, Shen CR, Li H, Sung L-Y, Wu M-Y, Hu Y-C (2016) CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942. Microb Cell Fact 15(1): 196.
[2] Yuste, L., Canosa, I., & Rojo, F. (1998). Carbon-source-dependent expression of the PalkB promoter from the Pseudomonas oleovorans alkane degradation pathway. Journal of bacteriology, 180(19), 5218–5226.