Difference between revisions of "Part:BBa R0061:Experience"
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BS_United_China 2023: we utilized this part to create our blue light suicide system, which is a system that will start when our engineering bacteria lost in the external environment is not exposed to blue light. This region of luxI promoter will be used in our design, where the EL222 binding region is located between −35 (TTGACA) and −10 (TATAAT) regions of the luxI promoter. In this way, once blue light is undetectable by EL222, the formation of EL222 dimers is terminated and the RNAP is allowed to bind back to the -35 to -10 region of the luxI promoter. This thereby activates the expression of the two types of CAS9 (CAS9-A and CAS9-R) and their sgRNA sequence (sgRNA-A and sgRNA-R). The gene editing process takes place after that, disrupting two of the most crucial parts of the bacterial cell’s metabolic pathways, energy and protein production, which eventually cause the death of bacteria.  
 
BS_United_China 2023: we utilized this part to create our blue light suicide system, which is a system that will start when our engineering bacteria lost in the external environment is not exposed to blue light. This region of luxI promoter will be used in our design, where the EL222 binding region is located between −35 (TTGACA) and −10 (TATAAT) regions of the luxI promoter. In this way, once blue light is undetectable by EL222, the formation of EL222 dimers is terminated and the RNAP is allowed to bind back to the -35 to -10 region of the luxI promoter. This thereby activates the expression of the two types of CAS9 (CAS9-A and CAS9-R) and their sgRNA sequence (sgRNA-A and sgRNA-R). The gene editing process takes place after that, disrupting two of the most crucial parts of the bacterial cell’s metabolic pathways, energy and protein production, which eventually cause the death of bacteria.  
 
The detailed design is showcased below:
 
The detailed design is showcased below:
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[[File:Fig. 10 OFF State_Blue light suircide system.png]
+
 
 +
[[File:Fig. 10 OFF State_Blue light suicide system|700px|thumb|center|Fig 1. Off State occurred when blue light is absorbed by EL222. CAS9 is deactivated. The blue light suicide system is not complete and thus the engineering E. coli lives. ]]
  
 
===User Reviews===
 
===User Reviews===

Revision as of 05:31, 7 October 2023

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_R0061

BS_United_China 2023: we utilized this part to create our blue light suicide system, which is a system that will start when our engineering bacteria lost in the external environment is not exposed to blue light. This region of luxI promoter will be used in our design, where the EL222 binding region is located between −35 (TTGACA) and −10 (TATAAT) regions of the luxI promoter. In this way, once blue light is undetectable by EL222, the formation of EL222 dimers is terminated and the RNAP is allowed to bind back to the -35 to -10 region of the luxI promoter. This thereby activates the expression of the two types of CAS9 (CAS9-A and CAS9-R) and their sgRNA sequence (sgRNA-A and sgRNA-R). The gene editing process takes place after that, disrupting two of the most crucial parts of the bacterial cell’s metabolic pathways, energy and protein production, which eventually cause the death of bacteria. The detailed design is showcased below:

File:Fig. 10 OFF State Blue light suicide system
Fig 1. Off State occurred when blue light is absorbed by EL222. CAS9 is deactivated. The blue light suicide system is not complete and thus the engineering E. coli lives.

User Reviews

UNIQa6551c3ccd60fb87-partinfo-00000000-QINU

No review score entered. iGEM Tokyo_Tech 2010

In order to characterize R0061, Plux repression promoter, we constructed K395101 combining R0061 and K121013, which is a promoter-less gfp reporter (rbs-gfp-ter-ter) on pSB6A1 and used a fusion of PlacIq (I14032) to gfp (K121013) as a positive control and used promoterless gfp (K121013) as a negative control.

Overnight cultures of reporter strains grown at 37 °C containing appropriated antibiotics were diluted at least 1:100 and incubated at 37 °C as fresh cultures. After their OD590 reached 0.6, added 3OC6HSL. After 3 hours of induction, fluorescence intensity was measured with flow cytometry.

After 3 hours of induction by 3OC6HSL, the expression of GFP with 3OC6HSL dropped to 1/3 comparing with the expression without 3OC6HSL.

We confirmed that this promoter works correctly. (→[http://2010.igem.org/Team:Tokyo_Tech/Project/Artificial_Cooperation_System/lux_act_rep more information])


Tokyotech R0061 K395008 graph R0061.jpg


iGEM Chiba 2010

iGEM Chiba 2010

  • To characterize R0061 biobrick part, we inserted a GFP reporter (E0240) under R0061 to make K396012.
  • We tested whether the GFP expression is repressed by LuxR and 3OC6HSL.
  • Repression could not be observed; we believe this is because we incubated the culture too much until stationary phase(see results & discussion below).
  • Other experiment that uses log phase cell (the original paper and the results from iGEM Tokyo Tech above) reports that the repression occurs.

Details

Materials & Methods
  • plamisd used
    1. K396012 on pSB2K3 (parts which GFP is inserted under R0061)
    2. K396011 on pSB1A3 (constitutive LuxR generator)
    3. J09250 on pSB2K3 (GFP under lac promoter) as a positive control
    4. R0061 on pSB2K3 as a negative control
    • All plasmids sequence confirmed.
    • notes: pSB2K3 is known as a very-low-copy plasmid, only in a lacIq strain. In this experiment we used DH10B which does not have lacIq.
  • strain
    • DH10B
  • procedure
    • plasmid 1 and 2 was cotransformed into DH10B. (plasmid 2 and 3 was used as a positive control, 3 and 4 as a negative control)
    • Colonies were cultured overnight in LB media with appropriate antibiotics.
    • Overnight cultures were diluted to 1:100 and incubated in a fresh LB medium (containing 0 or 1 uM 3OC6HSL and antibiotics) at 37 ºC for 12 h.
    • OD600 was measured, and 200 µL of the cultures were used to measure GFP fluorescence with a fluorescence microplate reader(excitation 485 nm,emission 527 nm).
Results
Figure. Fluorescence of GFP under lux repression promoter with/without AHL. p.c. stands for positive control, n.c. stands for negative control. All biobricks are co-transformed with K396011 (constitutive luxR generator). Bar heights are normalized to OD600 and represent the averages of three replicates; error bars, standard deviations.
  • Repression were not observed in the condition we tested (see right Figure).

  • Egland and Greenberg [1] (the original paper of this promoter) reported a strong repression of "Plux repression promoter" using log phase cells.
  • The results from iGEM Tokyo Tech 2010 (above in this page) shows about 1/3 repression also using log phase cells.
  • Cox et al. [2] reported that the "Plux repression promoter"' does not work. This paper measured the cells in a stationary phase (cultured at 25 ºC for 18 h) as our team did (at 37 ºC for 12 h).

  • From these information, we believe that the reason why we couldn't observe the repression is because we performed the experiment using stationary phase cells, not log phase cells.
Reference

<biblio>

  1. Egland pmid=10633117
  2. Cox pmid=18004278

</biblio>



UNIQa6551c3ccd60fb87-partinfo-00000003-QINU