Team:SHSBNU China/Experiments

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Experiments

Reporting Module

In our design, the induction signal will be detected and stored in the plasmid DNA sequence of our genetically modified E. coli. The bacteria will be gathered from the capsule after it left human body and sent to lab for further quantitative analysis in order to represent the inflammation level in colon. Therefore, in our project, the fluorescence intensity will be the index that represents levels of gut inflammation.
Since then, we first needed to develop a standard quantitative measurement protocol, so we studied the following articles: Zong, Y et al (2017)[1] and Zhang, H. M. et al(2015).

We constructed our reporter circuit with sfgfp and pTac promoter.

Figure 1

Following reference, we determined the experimental procedure:
Flat-bottom 96-well plates and sealing film were used throughout the study. Bacteria harboring parts/circuits of interest were inoculated from plates to LB medium and grown overnight (8−12 h, 1000 rpm, 37 °C, mB100-40 Thermo Shaker). Ten microliters of each overnight culture was sequentially diluted into 130 μL of fresh medium twice; the total dilution fold was 196. After growing the diluted cultures for ∼3 h, we diluted the exponentially growing cultures 700-fold using fresh medium; the dilution process was as follows: 10 μL of cell culture is added to 130 μL of M9 medium, which is followed by diluting 3 μL of this into 147 μL. Then, cultivation continued (1000 rpm, 37 °C, mB100-40); atspecifictimepoints,a2−50μL aliquot of each culture was transferred to a new plate containing 200 μL of PBS with 2 mg/mL kanamycin preadded to terminate protein expression. For the time course of cell growth after 700-fold dilution, OD 600 was recorded using Varioskan Flash (Thermal Scientific); the time interval was 5 min.
The graph below shows our result.

Figure 2
GFP is composed of 11 β-sheets and 1 α-helix. These peptides are connected by ‘Loop’. The amino acids of the 7, 10, and 11 β-sheet (such as H148, S205, E222, etc.) are combined with amino acids on the α-helix to form a chromophore (Cro).

Figure 3, GFP structure, Chi-Yun Lin, et,al.[1]

Figure 4, chromophore structure of GFP, James A. J et,al.[2]
We planned to add stop codons to be repaired onto GFP gene. Considering that loops between β-sheet and chromophore (Cro) have different influence on fluorescent of GFP, we designed mutations in multiple places. The table above depicted our result’s expectation. Green indicates the expected GFP fluorescence intensity, and the darkening of color indicates the disappearance of GFP fluorescence.

Figure 5
We envisioned that the mutations of Loop regions would have small effects to the GFP fluorescence intensity. If the repair efficiency is relatively high, mutations of multiple loop regions can show the fluorescence intensity increased with repair (different levels of fluorescence).

Figure 6
Mutations in the Cro region may have a huge effect on GFP brightness. If the repair is not efficient, the termination mutation in one Cro region will help to show the repaired cells (fluorescent or non-fluorescent).

Figure 7
Figure 7 shows the result of GFP mutation. The Y-axis is the fluorescence intensity. X-axis is strain number. Each strain has a different mutation at different positionsof GFP gene. Group 1 is control group, and the GFP is wild-type. Among these result, group 23 has the lowest fluorescent intensity, meaning that repair of this GFPmut will show the most folds of increase in brightness of GFP. Therefore, we decided to test GFPmut222 in order to find out whether SCRIBE can effectively repair DNA.

Figure 8
Figure 8 shows the qualitative result of strain1 (GFPwt) and strain23 (GFPmut222) when spreaded on LB agar plate. Picture on left shows the normal brightness of GFP picture on right shows brightness of induced expression of GFPmut222, the fluorescent of GFP is significantly decreased after adding a stop codon at the 222 amino acid. (LB Agar, IPTG 1mmol/L, for both plates) camera exposure: 1/30s，F/1.8，ISO100

SCRIBE Design

Figure 9
BBa_K5316888: a compact, modular strategy for producing single stranded DNA (ssDNA) inside of living cells in response to a range of regulatory signals. These ssDNAs address specific target loci on the basis of sequence homology and introduce precise mutations into genomic DNA.
In our experiment, we looked up resources about ssDNAs and focused on a SCRIBE system. And then, we referenced SCRIBE to construct the plasmids of BBa_K5316888 repair system.

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.
On another plasmid (pSB4K5), a stop codons is introduced into gfp gene to obtain a mutant GFPoff protein which is incapable of fluorescing. When the mutant gfp gene is repaired via recombination with engineered ssDNA (GFP)ON, it can re-luminesce. However, we found that the efficiency of SCRIBE transformation was very low in the end.

Golden Gate: The plasmid image shown below is the SCRIBE repair system. The repaired fragments are connected to RNA msr through golden gate. Golden Gate assembly uses BsaI, a IIS type endonuclease with different recognition sequence and digestion sequence. In our design, the regoc seq is at flank position of DNA fragment, so that after digestion, the regoc seq is cutted off, remaining the sticky end.The sticky end on msr-msd sequence is designed to match the inserted DNA, so different repairing seq could be replaced by only one enzyme, one protocol, convenient!

Figure 10

Debugging SCRIBE

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.

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.

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 Figure5). 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.

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.

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.

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