Part:BBa_K3937011
BADE+Lysis
This will be our final version of device. But it haven't work will, and we are going on experiment.
Design
In order to simultaneously realize the expression of the degradation enzyme and the initiation of autolysis, we designed the following two protocols
1.1Conjured the aflatoxin degradation enzyme BADE to the P43 promoter. Because P43, as an integrated promoter in Bacillus subtilis, does not need to be added as inducer compared with T7 and lactose operon, so it will be safer when adding feed. The lytic protein (BsrG, lytC) and the lytic protein promoter (PyqfD, PmmgA) were conjugated to each other by pin-pair combination. Finally, the degradation enzyme part and the lyase part were simultaneously cloned into the pHT01 plasmid, as shown in the figure. It can simultaneously express aflatoxin degradation enzyme, and express lysate protein after a certain period of time, start autolysate to release aflatoxin degradation enzyme, and finally complete the degradation of aflatoxin in the feed.
1.2According to the investigation, the pAX01 plasmid can be integrated into the genome of Bacillus subtilis, so four combinations of 1 lysin and lysin promoter can be cloned into the pAX01 plasmid respectively, and then integrated into the genome after transfer into Bacillus subtilis. The BADE gene of aflatoxin degradation enzyme was cloned into pHT01 by connecting with the P43 promoter. Finally, the constructed plasmid was transferred into Bacillus subtilis integrating with lytic protein, as shown in the figure. The expression and autolysis of degradation enzymes were realized.
Build
We conjured P43 to BADE and PyqfD to lytC by fusion PCR, and cloned both of them into pHT01 plasmid by homologous recombination. The plasmid was then transferred into the prepared WB600 receptor cells.
Test
3.1Growth curve
We added WB600 to LB medium for culture and measured its growth curve. It was found that the growth curve of the experimental group was almost the same as that of the control group transferred into empty plasmid, and there was no obvious curve decline in the experimental group during the stable period (the growth curve is shown in FIG 1).
Figure 1: Growth curve 1 shows the control group transferred into empty plasmid. 2-4 were transferred to the experimental group with two target genes
3.2SDS-PAGE
Meanwhile, after 24 h, the bacterial solution was prepared into the bacterial fragmentation solution for SDS-PAGE electrophoresis. The results showed that there was no significant difference in protein expression level, indicating that the expression levels of degradation enzyme and lysate protein were not significantly increased (as shown in Figure 2).
Figure 2: Protein SDS-PAGE electrophoresis
M: protein marker; 1: The cell fragmentation supernatant in 1 mL of the control group after 24h culture; In experimental group 1, the supernatant of bacterial fragmentation was obtained in 1 mL bacterial solution after 24h culture. In experimental group 2, the supernatant of cell fragmentation was obtained in 1 mL bacterial solution after 24h culture. In experimental group 3, the supernatant of thallus fragmentation was obtained in 1 mL bacterial solution after 24h culture
Learn
After discussing the experimental results with our instructor, we found that it may be the ribosome binding site followed by the connection of the target gene, resulting in the ribosome unable to correctly bind and perform functions. This resulted in no significant difference in the expression levels of degraded and lysed proteins.
Redesign
5.1Redesign the number of bases between ribosome binding sites and genes. To ensure that ribosomes bind properly and perform translation functions.
5.2Determine what site the lytic protein gene is connected to on pAX01, clone the target gene into pAX01 by homologous recombination method, and then transfer it into WB600.
5.3Determine the growth curve of the expression host obtained by the two methods, compare the differences between the two methods, and select the better method
5.4The combination of the remaining three lysins and lysin promoters was selected according to method 3 to construct expression host. The growth curve was also measured. Select the best self-cracking combination.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 497
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 497
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1040
Illegal BamHI site found at 82
Illegal XhoI site found at 935 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 497
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 497
Illegal AgeI site found at 254 - 1000COMPATIBLE WITH RFC[1000]
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