Part:BBa_K2556051

f1-AraC-Pbad-lysis-f2

We put AraC-Ara Promoter in front of the lysin gene from phage, and then we use CRISPR/Cas9 technology to turn the construction of AraC-Ara Promoter-Lysis into the genome of E.coli MG1655 wild strain. This part consists of fragments from the E.coli MG1655 genome, AraC-AraPromoter and lysin genes from phages.

Usage and Biology

In order to achieve cell lysis, we usually express the lysin gene. Then we find the part BBa_K2277000, which constructed by 2017 iGEM team ZJUT-China. They construct the lysinene in the plasmid. But in most cases, resistance genes will be added to plasmids in order to screen transformants. So we choose to construct the lysingene directly into the genome,for two advantages. First, it can reduce the number of plasmids that need to be transformed in the engineering bacteria, which can reduce the additional metabolic pressure. Second, it can avoid the addition of new resistance. We added the E.coligenome homology arm to both ends of this part. The genomic homology arms at both ends of our part allow us to insert this part into the non-metabolic pathway on the E.coli genome.

Characterize

We first built this part on plasmid T vector. To verify whether the expressed lysin protein has the function of cell lysis, we used E.coli MG1655 as a chassis. we transformed this plasmid into MG1655 and characterized the function of the lysin gene based on the growth curve of the bacteria. 

When the function of lysin protein was verified, we started to construct this part in the genome of E.coil MG1655 by using CRISPR/Cas9 system.

Experimental Results

Before genome editing, we first tested the cleavage function of the part containing the genomic homology arm on the plasmid. Our part is built on the T vector. The result is shown in Fig 1:

Fig.1 Growth curve

Our results show that when we added arabinose(the concentration is 10mM) to inducelysin gene expression, the OD₆₀₀ value of the bacterial culture was significantly lower than that of the control group without arabinose induction. And comparing with adding arabinose after 4.5 h of culture, when we added arabinose at the beginning, the OD₆₀₀ is lower.
Our vector experiments have initially confirmed that the expression of the lysin gene is effective for cell lysis. Based on the results of this experiment, we inserted the lysis part into the E.coli genome by CRISPR/Cas technology. In order to obtain a transformant that was successfully inserted the part into genome, we screened by plate streaking, and the experimental results are shown in Fig 2:

Fig.2 Result of plate streaking

We selected 34 transformants for plate streaking, and 5 of them showed lysis effects on arabinose containing plates. They were probably the strain we have insert lysis part into genome. And finally we got a strain with a significant lysis effect after arabinose induction, and we named it E.coli MG1655-Lysis. You can see the result in Fig. 3.

Fig.3 E.coli MG1655-Lysis cultured in tubes

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2200
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2826
  Illegal NgoMIV site found at 3636
  Illegal AgeI site found at 2035
  Illegal AgeI site found at 4001
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 2017
  

Contribution

• Group: [http://2019.igem.org/Team:UESTC-China iGEM Team UESTC-China 2019]
• Author: Jing Yao, Yingying Cao
• Summary: Characterization of the effect of lysin on the growth of Escherichia coli DH5α and BL21(DE3) strains by growth curve

Characterization from iGEM19-UESTC-China

We obtained the plasmid vector containing this part from 2019 iGEM team ZJUT-China. We transformed this plasmid into DH5α and BL21 (DE3) and mapped the corresponding growth curves to characterize the function of this part.In addition, the method of slab coating is used to further characterize the bactericidal effect of lysin.

Experiments Results

Growth curves

Fig. 1. OD600 curve of DH5α. The experimental data was derived from the average of two replicate experiments (three replicates per replicate). Each set of experimental data was separated by one hour.

Fig. 2. OD600 curve of BL21(DE3). The experimental data was derived from the average of two replicate experiments (three replicates per replicate). Each set of experimental data was separated by one hour.

Our experimental data showed that the growth of both host bacteria (E. coli DH5α and E. coli BL21) was strongly inhibited when the inducer arabinose (final concentration 10mM) was added to induce lysin gene expression. The bacterial density (OD600 value) of DH5α was changed from 0.38 at 5 h to 0.14 at 8 h, a decrease of 0.24 (Fig. 1). The bacterial density (OD600 value) of BL21 (DE3) decreased from 0.32 at 4 h to 0.06 at 7 h, a decrease of 0.26 (Fig. 2). At the same time, our data also showed that the lysin protein cleaves the E.coli BL21(DE3) for about 5h. When the expression is more than 5h, E.coli BL21(DE3) re-grows at a certain rate. This phenomenon suggests that when using lysin protein to control the growth of BL21, special attention should be paid to the processing time and additional treatments should be added.

Coating plate

Fig. 3. Regions 1, 2 and 3 are the control groups (E.coli DH5α with no lysin plasmid). Regions 4, 5 and 6 are experimental groups (E.coli DH5α with lysin plasmid).

Fig. 4. Regions 1, 2 and 3 are the control groups (E.coli BL21 with no lysin plasmid). Regions 4, 5 and 6 are experimental groups (E.coli BL21 with lysin plasmid).

The results of the coating plate experiment (Fig. 3 and Fig. 4) showed that the number of colonies in the blank group was significantly higher than that in the experimental group, so the lysin had significant bactericidal effects on both E.coli DH5α and BL21(DE3). The final concentration of ara is 10mM.

In our collaborations, we discussed with iGEM team ZJUT-China 2019(They used this lysin gene in their project.), then they decided to repeat our experiments. The repeated results are listed below by team ZJUT-China 2019. (See more details at https://2019.igem.org/Team:UESTC-China/Collaborations.)

Contribution

• Group: [http://2019.igem.org/Team:ZJUT-China iGEM Team ZJUT-China 2019]
• Author: Weiyan Jiang，Yanqiu Liu，Wei Zhang
• Summary: Characterization of the effect of lysin on the growth of E. coli DH5ɑ and BL21 strains by growth curve.

Characterize from ZJUT-iGEM

The lysis gene was constructed by ZJUT-China team in 2017. In this year, we transferred the plasmid into different host cells and measured the expression of this system 1. We transferred the plasmid containing lysis gene into E. coli DH5ɑ host cells and mapped the corresponding growth curves (Fig. 1). 2. We transferred the plasmid containing lysis gene into E. coli BL21 host cells and drew the corresponding growth curve (Fig. 2). 3. We used the coated plate method to verify the expression of cleavage gene. The bacteria was inoculated into medium containing arabinose and medium without arabinose (Fig. 3 and Fig. 4).

Growth curves

                        Fig. 1 Growth Curve of E. coli DH5ɑ                                                     Fig. 2 Growth Curve of E. coli BL21

Coating plate

Fig.3 Results of DH5α coated plates without Ara. The experimental data was derived from three parallel experiments

Fig.4 Results of DH5α coated plates with 27 mM Ara. The experimental data was derived from three parallel experiments

Experimental results

From our data, it can be found that the lysis gene is stably expressed in the E. coli DH5ɑ host cells, but occurs leakage expression in E. coli BL21 host cells. Our results show that when the arabinose operon was added to induce lysis gene expression, the growth of both E. coli DH5α and BL21 were significantly inhibited, with their OD₆₀₀ values decreased significantly. In E. coli DH5α host cells, the OD₆₀₀ decreased from 0.232 after 4 hour cultivation to 0.088 after 6 hour cultivation, and the difference was 0.144 (Fig. 5). The OD600 in E. coli BL21 host cells decreased from 0.278 after 4 hour cultivation to 0.093 after 6 hour cultivation, and the difference was 0.185. However, when the host cell was E. coli BL21, the OD₆₀₀increased significantly after 4-5 hours of lysis gene expression. The OD₆₀₀ increased from 0.071 in the 8th hour to 0.214 in the 10th hour, the difference was 0.143 (Fig. 6). The results of the coating plate experiment also showed that the lysis genes are very effective.These results suggest that in E. coli BL21 host cells, leakage of expression occurs after 4-5 hours of lysis gene expression. Therefore, we should pay attention to the time period which the lysis gene works and complete the discarding of the lysis gene during this time. By changing the growth environment, overacidity and overheating would be adopted to make the bacteria no longer have reproductive capacity.

Fig. 5 OD₆₀₀ of E. coli DH5α
Fig. 6 OD₆₀₀ of E. coli BL21

Reference

[1]Jiang W, Bikard D, Cox D, et al. CRISPR-assisted editing of bacterial genomes[J]. Nature Biotechnology, 2013, 31(3):233-239.
[2]Doench J G, Fusi N, Sullender M, et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9[J]. Nature Biotechnology, 2016, 34(2):184-191.
[3]Wang G, Lu X, Zhu Y, et al. A light-controlled cell lysis system in bacteria.[J]. Journal of Industrial Microbiology & Biotechnology, 2018:1-4.
[4]Park T, Struck D K, Dankenbring C A, et al. The Pinholin of Lambdoid Phage 21: Control of Lysis by Membrane Depolarization[J]. Journal of Bacteriology, 2007, 189(24):9135.