Difference between revisions of "Part:BBa R0061:Experience"
(→Details) |
|||
(11 intermediate revisions by 3 users not shown) | |||
Line 4: | Line 4: | ||
===Applications of BBa_R0061=== | ===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 CAS9 and its sgRNA sequence. 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: | ||
+ | |||
+ | <html> | ||
+ | <img src="https://static.igem.wiki/teams/4897/wiki/safety/fig-10-off-state-blue-light-suircide-system.png" width="500" height="auto">/ | ||
+ | </html> | ||
+ | |||
+ | <html> | ||
+ | <img src="https://static.igem.wiki/teams/4897/wiki/safety/fig-11-on-state-blue-light-suicide-system.png" width="500" height="auto">/ | ||
+ | </html> | ||
===User Reviews=== | ===User Reviews=== | ||
Line 40: | Line 50: | ||
|width='60%' valign='top'| | |width='60%' valign='top'| | ||
====iGEM Chiba 2010==== | ====iGEM Chiba 2010==== | ||
− | *To | + | *To characterize R0061 biobrick part, we inserted a GFP reporter ([[Part:BBa_E0240|E0240]]) under R0061 to make [[Part:BBa_K396012|K396012]]. |
*We tested whether the GFP expression is repressed by LuxR and 3OC6HSL. | *We tested whether the GFP expression is repressed by LuxR and 3OC6HSL. | ||
− | *Repression could not be observed | + | *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. | *Other experiment that uses log phase cell (the original paper and the results from iGEM Tokyo Tech above) reports that the repression occurs. | ||
+ | |||
====Details==== | ====Details==== | ||
=====Materials & Methods===== | =====Materials & Methods===== | ||
*plamisd used | *plamisd used | ||
− | *#[[Part:BBa_K396012|K396012]] on pSB2K3 (parts which GFP is inserted | + | *#[[Part:BBa_K396012|K396012]] on pSB2K3 (parts which GFP is inserted under R0061) |
*#[[Part:BBa_K396011|K396011]] on pSB1A3 (constitutive LuxR generator) | *#[[Part:BBa_K396011|K396011]] on pSB1A3 (constitutive LuxR generator) | ||
*#[[Part:BBa_J09250|J09250]] on pSB2K3 (GFP under lac promoter) as a positive control | *#[[Part:BBa_J09250|J09250]] on pSB2K3 (GFP under lac promoter) as a positive control | ||
Line 60: | Line 71: | ||
**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. | **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. | ||
**OD<sub>600</sub> 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). | **OD<sub>600</sub> 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===== | =====Results===== | ||
[[Image:Chiba R0061.png|right|300px|thumb|'''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 OD<sub>600</sub> and represent the averages of three replicates; error bars, standard deviations. ]] | [[Image:Chiba R0061.png|right|300px|thumb|'''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 OD<sub>600</sub> and represent the averages of three replicates; error bars, standard deviations. ]] |
Latest revision as of 01:53, 10 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 CAS9 and its sgRNA sequence. 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:
/
/
User Reviews
UNIQdb63ae3e0946def1-partinfo-00000002-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. |
iGEM Chiba 2010 |
iGEM Chiba 2010
DetailsMaterials & Methods
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.
Reference<biblio>
</biblio> |
UNIQdb63ae3e0946def1-partinfo-00000005-QINU