In order to make our engineering bacteria secrete CotAGold laccase only when there’s Aflatoxin B1 (AFB1) in the environment, we have designed the following plasmids, acted as an organic system, which completed the degradation of AFB1 together.
pGG (Nano-antibody_pACYCDuet-1)
Verification primer:
Primer 5: CGCCAATCAGCAACGACT
Primer 6: CGTGTTCTTATCGTTATCCC
In the context of plasmid pACYCDuet-1, this particular plasmid is of paramount importance, as it is responsible for the expression of the genetic material. This particular component comprises a nano-antibody that is capable of binding specifically to AFB1, coupled with a Gauss luciferase that is linked to the nano-antibody via a linker. Upon the nano-antibody binding to AFB1, a conformational change occurs, resulting in the oxidation of the substrate by the Gauss luciferase and the subsequent emission of blue light. The blue light has the ability to restore the activity of T7 RNA polymerase, thereby facilitating the subsequent expression of laccase.
pVVD (VVD_pCDFDuet-1)
Verification primer:
Primer 3: CGGCATACTCTGCGACAT
Primer 4: CGGTTGAAACATCGGACTA
Before the experiment, we have searched that the lactobacillus rhamnosus doesn’t have T7 RNA polymerase. So we used the extra T7 RNA polymerase to control the expression of the CotAGold laccase.
We divided the T7 RNA polymerase in to two parts, each of whom have been linked with a VVD structural domain, when the VVD structural domain was illuminated by blue light, two VVD structural domain will combine together so the two part T7 RNA polymerase will combine into a complete T7 RNA polymerase. So the CotAGlod and GFP protein which use T7 promoter only expressed when blue light is in the environment.
pCG (CotAGold+GFP_pET-30b(+))
Verification primer:
Primer 1: TCAGGAACGCACCATTAG
Primer 2: CGTCTATCAGGGCGATGG
We selected the CotAGold laccase as a “crusher”, which can specifically degrade AFB1.
The CotAgold gene used the T7 promoter, so it will only be express when the T7 RNA polymerase is active. (when the VVD structural domains were combined together because of the blue light).
pBS-MazF (PGlu+MazF_pBS(KS+))
Primer 7: TCCCATTCGCCATTCAGG
Primer 8: ATCTGCTAAGGCAACACC
We designed the suicide system by using a glucose promoter to control the expression of the MazF RNase. The glucose promoter will be active when the environment is in low glucose level, then it will start the transcription of the MazF gene, degrade the bacteria’s RNA and finally cause the death of the bacteria.
So those are the four plasmids we used. Eventually, they will form a fine laccase synthesis pathway that can efficiently degrade AFB1，As shown in the following picture.
