Difference between revisions of "Part:BBa K4593001"
 
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__NOTOC__
 
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<partinfo>BBa_K4593001 short</partinfo>
 
<partinfo>BBa_K4593001 short</partinfo>
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This part is the ORF coding of SPN1S_LysRZ
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This part contains the ORF coding for Rz-like spanin and SPN1S endolysin, connected with an RBS. It could be inserted to the place for ORF of a gene circuit, and must have full function as a whole.
  
 
===Usage and Biology===
 
===Usage and Biology===
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Spn1s_LysRZ is an protein complex that is consists of holin, endolysin and two Rz/Rz1-like protein that act together to exhibit high lytic activity against E. coli and Salmonella Typhimurium [1].
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Spn1s_LysRZ is a protein complex that consists of holin, endolysin and two Rz/Rz1-like spanin that act together to exhibit high lytic activity against E. coli and Salmonella Typhimurium [1].
  
  
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==Team:BNDS-China 2023==
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==Team: BNDS-China 2023==
 
Our project intends to develop an in-vivo elimination capsule targeting S. aureus. Spn1s_LysRZ is used to lyse E. coli and release the S. aureus targeting endolysin it expressed.  
 
Our project intends to develop an in-vivo elimination capsule targeting S. aureus. Spn1s_LysRZ is used to lyse E. coli and release the S. aureus targeting endolysin it expressed.  
  
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To characterize the lytic efficiency of Spn1s_LysRZ, we designed plasmid Spn1s_LysRZ-pET28a (assembled by Genscript) (Fig. 1), which allows Spn1s_LysRZ to be expressed under the presence of IPTG.  
 
To characterize the lytic efficiency of Spn1s_LysRZ, we designed plasmid Spn1s_LysRZ-pET28a (assembled by Genscript) (Fig. 1), which allows Spn1s_LysRZ to be expressed under the presence of IPTG.  
  
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/figure-19.png" width="700" height="auto"/>
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Fig. 1  The plasmid map of Spn1s_LysRZ-pET28a
 
Fig. 1  The plasmid map of Spn1s_LysRZ-pET28a
  
 
====Examining lysing ability using spectrophotometer====
 
====Examining lysing ability using spectrophotometer====
  
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To get a view of the self-lytic process at different expression levels over time, E. coli BL21 (ED3) with Spn1s_LysRZ-pET28a was shacked overnight and then diluted 500-fold into fresh LB medium (K+). The culture is separated into 4 group of different sterilized tubes, 5 ml each, after its OD 600 reaches 1.1. Various amount of 1 M IPTG is added to each tube subsequently, resulting designated final concentration (0mM, 0.01mM, 0.1mM, 1mM). The OD 600 of each group is recorded (with 3 repeats each time) at 15, 30, 60, 120 and 240 minutes after IPTG is added (Fig. X2).
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To get a view of the self-lytic process at different expression levels over time, E. coli BL21 (ED3) with Spn1s_LysRZ-pET28a was shaken overnight and then diluted 500-fold into fresh LB medium (K+). The culture is separated into 4 groups of different sterilized tubes, 5 ml each, after its OD 600 reaches 1.1. Various amount of 1 M IPTG is added to each tube subsequently, resulting designated final concentration (0mM, 0.01mM, 0.1mM, 1mM). The OD 600 of each group is recorded (with 3 repeats each time) at 15, 30, 60, 120 and 240 minutes after IPTG is added (Fig. X2).
 
   
 
   
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/figure-20.png" width="700" height="auto"/>
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Fig. 2  OD 600 of E. coli with plasmid Spn1s_LysRZ-pET28a under different IPTG concentrations with respect to time. (error bar is too small to be visible)
  
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Fig. 2  OD 600 of E. coli with plasmid Spn1s_LysRZ-pET28a under different IPTG concentration with respect to time. (error bar is too small to be visible)
 
  
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As the increase in protein concentration is not instant, all 4 groups show an increase in OD 600 during the first 15-minute interval. However, the groups with inducer show a significant decrease in growth speed and even a decrease in OD600 after 30 minutes or longer of induction compared with the blank group. Although protein overexpression could result in slower growth of bacteria, it could not lead to such a significant decrease in bacteria density in a short amount of time. Therefore, the result reveals that Spn1s_LysRZ is actively lysing E. coli.
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As the increase in protein concentration is not instant, all 4 groups show an increase in OD 600 during the first 15 min interval. However, the groups with inducer shows significant decrease in growth speed and even decrease in OD600 after 30 minutes or longer of induction compared with the blank group. Although protein overexpression could result in slower growth of bacteria, it could not lead to such a significant decrease in bacteria density in a short amount of time. Therefore, the result reveals that Spn1s_LysRZ is actively lysing E. coli.
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====visualizing lysing ability by plate spreading====
 
====visualizing lysing ability by plate spreading====
  
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To better visualize the result, after 240 min of induction, each group of culture is diluted 300-fold and 200 ul of each diluted bacteria culture is spreaded on K+ LB plates. The colony density on each plate after growing overnight is consistent with the relationship generated from OD 600 measurement. Overall, the result reveals a positive correlation between Spn1s_LysRZ expression level and lysing speed of E. coli, proving that this part is working successfully.
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To better visualize the result, after 240 min of induction, each group of culture is diluted 300-fold and 200 ul of each diluted bacteria culture is spread on K+ LB plates. The colony density on each plate after growing overnight is consistent with the relationship generated from OD 600 measurement. Overall, the result reveals a positive correlation between Spn1s_LysRZ expression level and lysing speed of E. coli, proving that this part is working successfully.
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Fig. 3  Overnight LB agar plate of E. coli with plasmid Spn1s_LysRZ-pET28a induced with different IPTG concentration for 240 min.
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A) 0.01mM IPTG.      B) 0.1 mM IPTG.      C) 1 mM IPTG.      D) 0 mM IPTG
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<figure>
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<p style="text-align:center;"><img src="https://static.igem.wiki/teams/4593/wiki/result/figure-21.png" width="700" height="auto"/>
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</figcaption>
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</figure>
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</html>
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Fig. 3  Overnight LB agar plate of E. coli with plasmid Spn1s_LysRZ-pET28a induced with different IPTG concentrations for 240 min.
  
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A)  0.01mM IPTG 
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B)  0.1 mM IPTG
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C)  1 mM IPTG. 
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D)  0 mM IPTG
  
  

Latest revision as of 03:30, 11 October 2023


Spn1s_LysRZ

This part contains the ORF coding for Rz-like spanin and SPN1S endolysin, connected with an RBS. It could be inserted to the place for ORF of a gene circuit, and must have full function as a whole.

Usage and Biology

Spn1s_LysRZ is a protein complex that consists of holin, endolysin and two Rz/Rz1-like spanin that act together to exhibit high lytic activity against E. coli and Salmonella Typhimurium [1].


Team: BNDS-China 2023

Our project intends to develop an in-vivo elimination capsule targeting S. aureus. Spn1s_LysRZ is used to lyse E. coli and release the S. aureus targeting endolysin it expressed.

Characterization of lytic activity when expressed in E.coli

To characterize the lytic efficiency of Spn1s_LysRZ, we designed plasmid Spn1s_LysRZ-pET28a (assembled by Genscript) (Fig. 1), which allows Spn1s_LysRZ to be expressed under the presence of IPTG.

Fig. 1 The plasmid map of Spn1s_LysRZ-pET28a

Examining lysing ability using spectrophotometer

To get a view of the self-lytic process at different expression levels over time, E. coli BL21 (ED3) with Spn1s_LysRZ-pET28a was shaken overnight and then diluted 500-fold into fresh LB medium (K+). The culture is separated into 4 groups of different sterilized tubes, 5 ml each, after its OD 600 reaches 1.1. Various amount of 1 M IPTG is added to each tube subsequently, resulting designated final concentration (0mM, 0.01mM, 0.1mM, 1mM). The OD 600 of each group is recorded (with 3 repeats each time) at 15, 30, 60, 120 and 240 minutes after IPTG is added (Fig. X2).

Fig. 2 OD 600 of E. coli with plasmid Spn1s_LysRZ-pET28a under different IPTG concentrations with respect to time. (error bar is too small to be visible)


As the increase in protein concentration is not instant, all 4 groups show an increase in OD 600 during the first 15-minute interval. However, the groups with inducer show a significant decrease in growth speed and even a decrease in OD600 after 30 minutes or longer of induction compared with the blank group. Although protein overexpression could result in slower growth of bacteria, it could not lead to such a significant decrease in bacteria density in a short amount of time. Therefore, the result reveals that Spn1s_LysRZ is actively lysing E. coli.

visualizing lysing ability by plate spreading

To better visualize the result, after 240 min of induction, each group of culture is diluted 300-fold and 200 ul of each diluted bacteria culture is spread on K+ LB plates. The colony density on each plate after growing overnight is consistent with the relationship generated from OD 600 measurement. Overall, the result reveals a positive correlation between Spn1s_LysRZ expression level and lysing speed of E. coli, proving that this part is working successfully.

Fig. 3 Overnight LB agar plate of E. coli with plasmid Spn1s_LysRZ-pET28a induced with different IPTG concentrations for 240 min.

A) 0.01mM IPTG

B) 0.1 mM IPTG

C) 1 mM IPTG.

D) 0 mM IPTG



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 391
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 890
    Illegal AgeI site found at 1028
  • 1000
    COMPATIBLE WITH RFC[1000]