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Revision as of 05:42, 20 October 2021

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Selection Marker Parts | My Site

Selection Marker Parts

Biobricks to revolutionize the Safety in the Lab

The design of these parts was made thinking about a novel selection marker and a modified strain of E. coli we will use to substitute the use of antibiotics in genetic engineering protocols. The idea is to use this parts to generate a knockout strain from E. coli DH5⍺ genes oxyR, gldA and glpK and substitute with aldO, mRFP and PuDHT genes. We also designed a part that forms a plasmid and acts as a selection cassette. AldO will convert glycerol to peroxide and D-glyceric acid, katE will convert peroxide to water and oxygen and PuDHT will convert D-glyceric acid into pyruvate. We will briefly explore the functionality of each part, as well as key aspects of its design.

BBa_K3809001: Novel Selection Cassette katE

T--TecCEM--PartsFig5.png

Figure 1.

Map of BBa_K3809001. Created with Snapgene.

This part codes for the E. coli catalase gene katE which converts peroxide into water and oxygen. It is meant to be used with a pUC backbone we designed in which the part acts as a selection marker for the modified strain.  For more information, click on the part name.

BBa_K3809003: katE regulated by BBa_J23100

T--TecCEM--figura2.png

Figure 2.

Map of BBa_K3809003. Created with Snapgene

This Biobrick contains a katE gene with an RBS (BBa_B0030), spacer (BBa_B0040), terminator (BBa_B0010) and promoter (BBa_J23100). It was designed to be expressed as part of a plasmid, to act as a selection cassette for a modified strain of E. coli.

BBa_K3809004: aldO

T--TecCEM--fig3.png

Figure 3.

Map of BBa_K3809004. Created with Snapgene.

This sequence codes for the enzyme alditol oxidase from the organism Streptomyces coelicolor. It was designed as part of a novel selection method in which the enzyme converts glycerol into D-glycerate. 

BBa_K3809005: aldO + glpK homology regions

T--TecCEM--fig4.png

Figure 4.

Map of BBa_K3809005. Created with Snapgene

This part corresponds to the coding sequence of alditol oxidase. It is regulated by the BBa_J23100 promoter and has a BBa_B0030 RBS and an rrnB T1 terminator BBa_B0010. It also has a spacer between the promoter and the RBS (BBa_B0040). The sequence contains two homology regions for the gene glycerol kinase (glpK) of E. coli, which is a key enzyme in the glycerol metabolic pathway.

BBa_K3809006: PuDHT

T--TecCEM--fig5.png

Figure 5.

Map of BBa_K3809006. Created with Snapgene

This sequence codes for the enzyme dehydratase from the organism Paralcaligenes ureilyticus. It was designed as part of a novel selection method in which the enzyme converts D-glyceric acid into Pyruvate, which is a useful compound for bacteria.

BBa_K3809007: PuDHT + gldA homology regions

T--TecCEM--fig6.png

Figure 6.

 Map of BBa_K3809007. Created with Snapgene

This part corresponds to the coding sequence of alditol oxidase. It is regulated by the BBa_J23100 promoter and has a BBa_B0030 RBS and an rrnB T1 terminator BBa_B0010. It also has a spacer between the promoter and the RBS (BBa_B0040). The sequence contains two homology regions for the gene glycerol kinase (glpK) of E. coli, which is a key enzyme in the glycerol metabolic pathway.

BBa_K3809014: mRFP + oxyR homology regions + FLP recognition sequence

T--TecCEM--fig7.png

Figure 7.

Map of BBa_K3809014. Created with Snapgene

This sequence codes for the reporter protein mRFP. It was designed as part of a novel selection method in which the oxidative stress regulator gene (oxyR) is removed. mRFP will act as a reporter and will indicate if the knockout was successful or not. This gene will work alongside aldO, PuDHT and katE to develop a new selection mechanism which involves the knockout of genes gldA, glpK and oxyR to make E. coli susceptible to death by exposure to low concentrations of glycerol.

 

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