Difference between revisions of "Part:BBa K2447500:Experience"

 
Line 3: Line 3:
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
how you used this part and how it worked out.
 +
 +
In this construct, the photosensitive bacterial protein is constructively produced. In the presence of blue light, EL222 is activated via dimerisation and is able to bind upstream of the pLuxI promoter, thereby allowing the recruitment of RNA polymerase to activate expression of RFP. In the absence of blue light, EL222 does not dimerise and is unable to bind DNA; hence showing minimal RFP expression.
 +
 +
[[Image:blue5.png|thumb|center|800px| Figure 1: Under blue-light condition, EL222 is recruited upstream of the RNA polymerase binding region and recruits the polymerase for downstream expression of RFP. Figure is obtained from this [https://www.ncbi.nlm.nih.gov/pubmed/27353329 article.]]]
 +
 +
 +
====Usage and Characterisation====
 +
This part is inserted into pBbE8k backbone and subsequently characterized in E. coli 10Beta and MG 1655. These cells are grown in LB medium (with kanamycin) at 37 degrees to reach OD600 of 0.1 before blue light is administered for 7 hours. For every hour, RFP readings were obtained and cross-compared with the control (no blue light).
 +
[[Image:blue4.jpeg|thumb|center|400px| Figure 2: Incubator-shaker used in the blue light experiment.]]
 +
[[Image:blue3.jpeg|thumb|center|300px|Figure 3: Blue light being shone on one plate while no light condition was ensured with the covering of a black cloth.]]
 +
 +
 +
Strong RFP expression was shown from both cell types (under blue-light condition) as compared to the control condition where no light was administered. By the 6th hour onwards, RFP productions had tripled for both cell types when compared to the control condition.
 +
In short, this construct could prove useful for any group seeking to elucidate gene expression based on blue light as a physical stimulus. 
 +
 +
 +
[[Image:blue2.png|thumb|center|800px| Figure 4: MG 1655 under blue light and no light conditions.]]
 +
[[Image:blue1.png|thumb|center|800px|Figure 5: 10Beta under blue light and no light conditions.]]
  
 
===Applications of BBa_K2447500===
 
===Applications of BBa_K2447500===

Revision as of 11:08, 1 November 2017


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.

In this construct, the photosensitive bacterial protein is constructively produced. In the presence of blue light, EL222 is activated via dimerisation and is able to bind upstream of the pLuxI promoter, thereby allowing the recruitment of RNA polymerase to activate expression of RFP. In the absence of blue light, EL222 does not dimerise and is unable to bind DNA; hence showing minimal RFP expression.

Figure 1: Under blue-light condition, EL222 is recruited upstream of the RNA polymerase binding region and recruits the polymerase for downstream expression of RFP. Figure is obtained from this article.


Usage and Characterisation

This part is inserted into pBbE8k backbone and subsequently characterized in E. coli 10Beta and MG 1655. These cells are grown in LB medium (with kanamycin) at 37 degrees to reach OD600 of 0.1 before blue light is administered for 7 hours. For every hour, RFP readings were obtained and cross-compared with the control (no blue light).

Figure 2: Incubator-shaker used in the blue light experiment.
Figure 3: Blue light being shone on one plate while no light condition was ensured with the covering of a black cloth.


Strong RFP expression was shown from both cell types (under blue-light condition) as compared to the control condition where no light was administered. By the 6th hour onwards, RFP productions had tripled for both cell types when compared to the control condition. In short, this construct could prove useful for any group seeking to elucidate gene expression based on blue light as a physical stimulus.


Figure 4: MG 1655 under blue light and no light conditions.
Figure 5: 10Beta under blue light and no light conditions.

Applications of BBa_K2447500

User Reviews

UNIQ1471e2927b2099d1-partinfo-00000000-QINU UNIQ1471e2927b2099d1-partinfo-00000001-QINU