Team:XHD-Wuhan-B-China/Engineering
Engineering
oriC4
OriC4 refers to No.4 oriC binding site tested by Wiktor's group [1] which is proven to be functional of inhibiting replication initiation. When oriC is inhabited, E.coli cells can trigger neither replication initiation nor cell division.
We named our sgRNA that binds No.4 oriC binding site as “oriC4”. To test it, we used an old version of dCas9 plasmid where dCas9 was expressed by J23103, coupled with a CcaS-CcaR plasmid where oriC4 was expressed by PcpcG2-68 promoter (plasmids were provided by our host lab). These two plasmids were co-transformed into DH5a strain. Light-control experiment was performed in a 24-well plate adapted device. Overnight culture was diluted to 50% and then shaken at 37°C, 200rpm, for 2 hours under green light. The bacteria suspensions became clarified with cells deposited at the bottom and became floccus after shaking. This phenomenon indicates the expression of oriC4 forced cells to become filamentous and possibly died during shaking.
dicF
Construction of dicF expression system induced by arabinose promoter (BBa_K3921006)
To prove that small RNA (sRNA) DicF can indeed inhibit the cell division of MG1655, we constructed a circuit containing pBAD-dicF gene (Fig. 1), enabling sRNA DicF to be induced by arabinose promoter. In order to realize the function of dicF circuit, we constructed plasmid map of pBAD-dicF (Fig. 2).
To prove that small RNA (sRNA) DicF can indeed inhibit the cell division of MG1655, we constructed a circuit containing pBAD-dicF gene (Fig. 1), enabling sRNA DicF to be induced by arabinose promoter. In order to realize the function of dicF circuit, we constructed plasmid map of pBAD-dicF (Fig. 2).
Figure 1. Schematics of the dicF circuit. In the presence of L-arabinose, dicF expresses as sRNA DicF. DicF degrades the mRNA of ftsZ. Escherichia coli without protein FtsZ will not be able to form a division septum, causing cell to become filamentous, and cell division is inhibited.
Figure 2. Plasmid map of pBAD-dicF.
Usage and Biology
We transformed pBAD-dicF plasmid into MG1655. In order to ensure that dicF was successfully constructed on pBAD24 plasmid, primers for colony PCR were designed in upstream and downstream of dicF. The theoretical PCR product size is 336bp, and gel electrophoresis results showed that pBAD-dicF plasmid was successfully constructed (Fig. 3). After transforming pBAD-dicF into MG1655, when the culture medium OD600 was 0.3, it was induced with arabinose (0.2%) for 12 hours and then observed under microscope (40×). As shown in Fig. 4A, compared with the strain pBAD-dicF MG1655 without arabinose (Fig. 4B), most of the strains induced by arabinose were filamentous, indicating that the process of cell division was inhibited. We also calculated the relative length of MG1655 cells in terms of the pixels of the microscope photos, and the relative length of cells induced with L-arabinose increased by about 3 times (Fig. 5).
We transformed pBAD-dicF plasmid into MG1655. In order to ensure that dicF was successfully constructed on pBAD24 plasmid, primers for colony PCR were designed in upstream and downstream of dicF. The theoretical PCR product size is 336bp, and gel electrophoresis results showed that pBAD-dicF plasmid was successfully constructed (Fig. 3). After transforming pBAD-dicF into MG1655, when the culture medium OD600 was 0.3, it was induced with arabinose (0.2%) for 12 hours and then observed under microscope (40×). As shown in Fig. 4A, compared with the strain pBAD-dicF MG1655 without arabinose (Fig. 4B), most of the strains induced by arabinose were filamentous, indicating that the process of cell division was inhibited. We also calculated the relative length of MG1655 cells in terms of the pixels of the microscope photos, and the relative length of cells induced with L-arabinose increased by about 3 times (Fig. 5).
Figure 3. Gel electrophoresis of verification of recombinant plasmid pBAD-dicF.
Figure 4. Microscopic (40×) observation map of pBAD-dicF MG1655. (A) Induced by adding L-arabinose. (B) Do not add L-arabinose. (A) and (B) were cultured in 200rpm shaker at 37 ℃ for the same time.
Figure 5. Statistical diagram of MG1655 cell length (In pixels).
Reference
[1]. Wiktor J, Lesterlin C, Sherratt DJ, Dekker C. CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res. 2016 May 5;44(8):3801-10.
[1]. Wiktor J, Lesterlin C, Sherratt DJ, Dekker C. CRISPR-mediated control of the bacterial initiation of replication. Nucleic Acids Res. 2016 May 5;44(8):3801-10.