Part:BBa_K3156888

pLac Promoter-ssDNA[sfGFP(ON)]-Ec86-Beta

Design

We combined msd-msr cassette, Reverse Transcriptase Ec-86 and Beta recombinase together to create BBa_K3156888. Once msr starts transcribing, the msr-msd RNA folds into a secondary structure which can be recognized by RT protein (Reverse Transcriptase) using a conserved guanosine residue in the msr as a priming site to reverse transcribe the msd sequence and produce a hybrid RNA-ssDNA molecule called msDNA.

Figure 1.Circuit design of BBa_K3156888(sfgfp forward).

We created a stimualte gif to help iGEMers to know more about our design.

Figure 2.Stimulated reparing progress of K3156888.

Culturing and plating

For each experiment, the samples were separately inoculated in LB medium plus appropriate antibiotics and grown overnight at 37°C, 200 revolutions per minute (RPM) to obtain seed cultures. Unless otherwise noted,inductions were performed by diluting the seed cultures (1:1000) in 2 ml of prewarmed LB plus appropriate antibiotics with or without inducers followed by 24 hours incubation 30°C, 200 RPM.

Figure 3.Repairing result of BBa_K3156888(sfgfp forward).

Debug process

In the previous experiment, we found out that the 222# mutation of GFP on PSB4K5 plasmid could not be repaired successfully. The lumination of experimental group has no difference with the control group.Under this circumstance, we started our “debug” process.

Figure 4.Changing reporter gene.

First, we highly doubted that the plasmid pSB4K5, has too much copies inside the bacteria cell. So we decided to move the target gene from plasmid backbone to the bacteria genome. The genome has much lower copy number than pSB4K5 plasmid, making the repair of our target gene easier. From our last year’s iGem project, we’ve already have an E.coli strain that has csgA located on the genome. So we switched our target of repair from gfp on pSB4K5 to csgA on E.coli genome.

Figure 5.Trigger reparation.

The csgA produced biological membrane. Therefore we could not see the lumination of the bacteria. Meanwhile, it is not easy to introduce mutation on genome. Because we were not sure about the whether the function of the entire genome would be affected or not. In order to prevent the genome from being damaged and make our experiment easier, we decided to introduce synonymous mutation to the csgA. If the synonymous mutation was introduced successfully, the reparation would be done technically. As the figure5 shows ,we inserted synonymous mutation into msd region on the scribe plasmid. Then we transferred the plasmid into E.coli. We used 0.1mmol IPTG as inducer. Then the mutation would be introduced into csgA. Then they were incubated for 24h. After incubation, we spread bacterial on the plate. And let them grow. However, the results of DNA sequencing showed our mutation were not introduced into the genome.

After discovering that genome could not be repaired as well, we went back to each step to check. Then we found that the direction of inserted mutation was essential in reparation. Because the Beta Protein in BBa_K3156888 system would only insert the gene sequence when replication fork occurred (See Figure 6). If the direction of our mutation sequence was reversed, mutation would not be introduced into the okazaki fragment. Therefore, we changed the direction of mutation sequence.

Figure 6.Changing the Direction of Renovation Sequence.

As we can see in the graph, the previous design was the one on the top. Beta protein could not carry ssDNA to the replication fork and insert it.
Since genome could not work as well, we went back to repair gfp mutation on plasmid. We redid the experiment after changing the direction of gfp sequence. In case there were possibilities that BBa_K3156888 system has low repairing rate. We did the bacterial continued culture with dilution and spreading every day.Then the colonies with high florescence intensity were sent to do flow cytometry.

Figure 7.Plating result

The picture showed colonies with high florescence intensity. Then we sent those bacterial to do the Flow Cytometry. In case there were any false positive control group. The Flow Cytometry results are shown in the table below.

Figure 1.Repairing result of BBa_K3156888(sfgfp reverse).

Fortunately, we do have successful reparation as expected. Therefore the “debug” process for BBa_K3156888 system is necessary and successfully support our design.

Reference

[1]F. Farzadfard, T. K. Lu, Science 346,1256272 (2014). DOI: 10.1126/science.1256272

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 9
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1
  Illegal XhoI site found at 516
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]