Difference between revisions of "Part:BBa E0840:Experience"

(→‎User Reviews)
(→‎User Reviews)
 
(3 intermediate revisions by 2 users not shown)
Line 14: Line 14:
 
===User Reviews===
 
===User Reviews===
 
<!-- DON'T DELETE -->
 
<!-- DON'T DELETE -->
 +
{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
 +
<partinfo>BBa_E0840 AddReview 5</partinfo>
 +
<I>iGEM Dundee 2016</I>
 +
|width='60%' valign='top'|
  
 +
The function of this part was characterised in a new bile salt-sensing device. This is a really good composite part. We linked it to a promoter from the <i>acrRA</i> operon (<partinfo>BBa_K318514</partinfo>) and co-expressed it with the RamA transcriptional activator (<partinfo>BBa_K1962009</partinfo>). The new composite part (a bile salt reporter) (<partinfo>BBa_K1962010</partinfo>) was found to work, inducing GFP production under test conditions.
 +
 +
We also used this part to characterise the pH-sensitive <i>gadA</i> promoter (<partinfo>BBa_K1231001</partinfo>) to generate a new composite part called <partinfo>BBa_K1962013</partinfo>.
 +
 +
We also used this part to characterise another pH-sensitive promoter - that of <i>asr</i> (<partinfo>BBa_K1231000</partinfo>). The resultant new composite part is available at <partinfo>BBa_K1962014</partinfo>.
 +
 +
Overall, we endorse the use of this excellent composite part (<partinfo>BBa_E0840</partinfo>) for characterising promoter activity in an <i>E. coli</i> chassis. The RBS-GFP-T1-T2 design means much time is saved in cloning with a single ligation sufficient before experiments can begin.
 +
|}
 
{|width='80%' style='border:1px solid gray'
 
{|width='80%' style='border:1px solid gray'
 
|-
 
|-
Line 30: Line 44:
  
 
Fluorescence can be observed in bacteria exposed to small concentrations, as 0.01µg/ml and 0.02µg/ml, but has low intensity. Fluorescence levels presents medium intensity in the higher concentration as consequence of cell death. Fluorescence intensity increased more than 480% in 0.2µg/ml concentrations, compared to fluorescence intensity from 1µg/ml, even in cell growth reduction, demonstrating its efficiency to induce mer promoter.
 
Fluorescence can be observed in bacteria exposed to small concentrations, as 0.01µg/ml and 0.02µg/ml, but has low intensity. Fluorescence levels presents medium intensity in the higher concentration as consequence of cell death. Fluorescence intensity increased more than 480% in 0.2µg/ml concentrations, compared to fluorescence intensity from 1µg/ml, even in cell growth reduction, demonstrating its efficiency to induce mer promoter.
 +
 +
Check it out our [https://parts.igem.org/Part:BBa_K1355002:Experience Experience BBa_K1355002] in extended version and the [https://parts.igem.org/Part:BBa_K1355002:Design Desing] of the Hg biosensor!
 
|}
 
|}
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
Line 57: Line 73:
 
Our miniprep, double and single digests worked as expected. We used this biobrick for building two of our parts (<partinfo>BBa_K182100</partinfo> and '<partinfo>BBa_K182101</partinfo>). We experienced very satisfactory cloning with high efficiency of transformants. The sequencing was correct.  We discovered that all the bacteria containing this biobrick were releasing fluorescence even though there was no promoter upstream. The level of fluorescence observed was dependent on the plasmid copy number. We noticed that colonies displayed a visible, yellow-green colour, therefore selection was very easy.
 
Our miniprep, double and single digests worked as expected. We used this biobrick for building two of our parts (<partinfo>BBa_K182100</partinfo> and '<partinfo>BBa_K182101</partinfo>). We experienced very satisfactory cloning with high efficiency of transformants. The sequencing was correct.  We discovered that all the bacteria containing this biobrick were releasing fluorescence even though there was no promoter upstream. The level of fluorescence observed was dependent on the plasmid copy number. We noticed that colonies displayed a visible, yellow-green colour, therefore selection was very easy.
 
|}
 
|}
 +
 +
 
<!-- DON'T DELETE --><partinfo>BBa_E0840 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_E0840 EndReviews</partinfo>
  

Latest revision as of 11:14, 16 October 2016

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_E0840

BBa_E0840 is commonly used to quantify the function of transcriptional control devices.

igem2010 UT-Tokyo

We made an inverted version of this part, BBa_K313007, with the use of PCR.

GFP in the reverse direction are especially useful as outputs of genetic switches in which the coding regions of GFP are inverted by the recombinases.

User Reviews

•••••

iGEM Dundee 2016

The function of this part was characterised in a new bile salt-sensing device. This is a really good composite part. We linked it to a promoter from the acrRA operon (BBa_K318514) and co-expressed it with the RamA transcriptional activator (BBa_K1962009). The new composite part (a bile salt reporter) (BBa_K1962010) was found to work, inducing GFP production under test conditions.

We also used this part to characterise the pH-sensitive gadA promoter (BBa_K1231001) to generate a new composite part called BBa_K1962013.

We also used this part to characterise another pH-sensitive promoter - that of asr (BBa_K1231000). The resultant new composite part is available at BBa_K1962014.

Overall, we endorse the use of this excellent composite part (BBa_E0840) for characterising promoter activity in an E. coli chassis. The RBS-GFP-T1-T2 design means much time is saved in cloning with a single ligation sufficient before experiments can begin.

•••••

UFAM_Brazil 2014

We used this biobrick part to build a Hg biosensor! This part worked so well!

The graph represented on Figure 1 shows the fluorescence emitted by DH5-alpha transformed with BBa_K1355002 induced by different Hg concentrations in function of the time:

Bs2.png

Figure 1. GFP fluorescence intensity in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.

Fluorescence can be observed in bacteria exposed to small concentrations, as 0.01µg/ml and 0.02µg/ml, but has low intensity. Fluorescence levels presents medium intensity in the higher concentration as consequence of cell death. Fluorescence intensity increased more than 480% in 0.2µg/ml concentrations, compared to fluorescence intensity from 1µg/ml, even in cell growth reduction, demonstrating its efficiency to induce mer promoter.

Check it out our Experience BBa_K1355002 in extended version and the Desing of the Hg biosensor!

••••

Smelissali

Very visible even without UV excitation on plate and in solution after 24 hours incubation. Not as visible as Part:BBa_I13521 (mRFP), however.

•••••

Reshma Shetty

BBa_E0840 was successfully used to quantify the behavior of BBa_R0040, BBa_J45992 and BBa_J45994.

•••••

Aberdeen_Scotland 2009

Our miniprep, double and single digests worked as expected. We used this biobrick for building two of our parts (BBa_K182100 and 'BBa_K182101). We experienced very satisfactory cloning with high efficiency of transformants. The sequencing was correct. We discovered that all the bacteria containing this biobrick were releasing fluorescence even though there was no promoter upstream. The level of fluorescence observed was dependent on the plasmid copy number. We noticed that colonies displayed a visible, yellow-green colour, therefore selection was very easy.


UNIQ869a53a5be3605b9-partinfo-00000014-QINU

Characterization

Transcriptional control of GFP generator

All three transcriptional control devices were quantified using BBa_E0840.

Growth phase dependent transcriptional control devices
We successfully designed, constructed and tested transcriptional control devices for constitutive, stationary phase dependent and exponential phase dependent protein production (A-C). To test and verify function of our three transcriptional control devices, we assembled each control device with the GFP protein generator BBa_E0840 and monitored the fluorescence of E. coli cultures with each device over time. For each device, we plot the change in fluorescence per unit time (normalized GFP synthesis rate) versus the cell density (OD600nm) (D). The constitutive transcriptional control device produced a high GFP synthesis rate irrespective of cell density. The stationary phase transcriptional control device produced a low initial GFP synthesis rate which increased with culture cell density. The exponential phase transcriptional control device produced an initially high GFP synthesis rate which dropped off as cell density increased. Data shown are averages of triplicate measurements of cultures grown from three individual colonies of each device. Error bars are the standard deviation of the three individual cultures.