Part:BBa_K5335013
This part includes holin and PGHs.holin is a type of perforin, and PGHs is a phage-encoded peptidogl
This part includes holin and PGHs.Holin is a type of perforin, and PGHs is a phage-encoded peptidoglycan hydrolases, both of which are derived from Phage VB_Bsup-GOe1 and act against B.subtilis.PGHs can hydrolyze peptidoglycans in bacterial cell walls, while holin can perforate their membranes, and the two together can cause B.subtilis to lyse and die. In our project, the engineered bacteria will synthesize VLP particles, and in order to ensure the smooth release of these protein particles outside the cell, we designed an element for lytic bacteria that contains these two genes.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 907
Illegal XbaI site found at 101 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 907
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 907
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 907
Illegal XbaI site found at 101 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 907
Illegal XbaI site found at 101 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The use of holin and PGHs causes bacterial cleavage, which we hope will release the protein products synthesized in them.
Control of bacterial lysis by binding to operons
We combined two gene sequences with a terminator of B.subtilis,and SpoVG RBS.These elements were shown to be usable in B.subtilis. We then reprogrammed the sequence into a pHT315 plasmid with xylose operon and induced expression using D-xylose.We introduced the recombinant pHT315 into B. Subtilis by electrocution.
Characterization
Agarose gel electrophoresis
The success of transformation was preliminatively confirmed by PCR of the transformed B.subtilis colony.
M: DL1000 DNA Marker; 1-6: Colony PCR bands for B.subtilis168 Top 6
Figure 1. Agarose gel electrophoresis validation of PCR results of B.subtilis168 colonies. The first lane was loaded with DL1000 DNA ladder.Sizes were marked on the image. We chose 2× Magic Green Taq SuperMix.It contains DNA Polymerase, dNTP, and an loading buffer system, which allows amplification by adding only primers and templates,the product can be electrophoretic directly after PCR,simplify the experimental process and improve the repeatability of the results. The PCR reaction consisted of 15 μL 2 × Magic Green Taq SuperMix, 1.5 μL forward primer (3.75 mM), 1.5 μL reverse primer (3.75 mM), 11 μL H2O and 1 μL colony.
Preliminary lysis verification
To quickly determine if our circuit works, we performed an extremely simple pre-experiment on the resulting strains, which intuitively and effectively reflected the action of the cleavage circuit.
Figure 2. D-xylose induced lysis pre-experiment.The bacterial solution of the same recombinant strain was divided into two parts, one part was added with 60 mM/L D-xylose (left in the figure), and the other part was not treated (right in the figure), and then cultured in a shaking bed at 37℃ overnight.
It can be seen that the bacterial solution in the D-xylose induced group was basically clarified, while the bacterial solution in the non-xylose group was very cloudy. This basically means that our circuit works.
Formal cracking verification (growth curve)
To further confirm the cleavage efficiency, we designed a formal experiment in which one group provided 60 mM/L D-xylose induction and the other group did not provide D-xylose, and the OD600nm value of the bacterial solution was continuously measured.
Figure 3. D-xylose induced lysis formal-experiment.The bacterial solution of the same recombinant strain was divided into two parts, one part was added with 60 mM/L D-xylose (Treatment), and the other part was not treated (Control),All bacteria were cultured in 10mL bacterial vials and the OD600 value was measured every 40 minutes for 8 consecutive times.
The lysis circuit plays a certain role in the D-xylose induction, but the effect is not obvious.
Formal cracking verification (hypoxia)
In order to reflect the function of the cracking circuit more directly, combined with the experience of the pre-experiment, we redesigned a new experimental condition. We compared common B.subtilis168 with transformed B.subtilis168, and induced them with different concentrations of D-xylose, and all the bacteria were treated in sealed membrane coated, oxygen-deprived culture bottles.
Figure 4. D-xylose induced lysis formal-experiment(hypoxia).D-xylose induced lysis formal-experiment(hypoxia).We've prepared two parts of solution,one part were normal B. Subtilis168 (Control),another part were transformed B. Subtilis168 (Treatment).Then they were added with different concentrations of D-xylose as shown in the figure, sealed with a breathable film, and cultured for 12 hours, after which the OD600 value was measured.
During this step, the inverting bacteria induced by D-xylose showed cleavage, while the non-inverting bacteria were almost unaffected. At the same time, the higher the concentration of induced D-xylose, the better the growth of recombinant bacteria. We analyzed that this may be because xylose itself is also the energy material of B.subtilis, and to a certain extent, high concentration of xylose promotes their growth.
Reference
Willms IM, Hertel R. Phage vB_BsuP-Goe1: the smallest identified lytic phage of B.subtilis. FEMS Microbiol Lett. 2016 Oct;363(19):fnw208. doi: 10.1093/femsle/fnw208
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