Difference between revisions of "Part:BBa K2447014"

Latest revision as of 08:28, 1 November 2017

IPTG-inducible temperature-sensitive system with GFP reporter

In the absence of IPTG, LacI repressor will bind to pLac promoter and inhibit further downstream expression. pLac promoter is attached upstream of the TlpA36 protein coding sequence to control the amount of TlpA36 protein expression. When IPTG is present and under high temperatures (above 36 degree Celsius), the temperature system will be "ON" and GFP is expressed. Under low temperatures (below 36 degree Celsius), the pTlpA will be repressed, which will stop GFP expression.

Usage and Characterisation

For more detailed experimental results, do visit our [http://2017.igem.org/Team:NUS_Singapore/Experiments experimental page].

This part is inserted into pBbE2k backbone and subsequently characterised in E. coli 10beta. These cells are grown in LB medium (with kanamycin) at 30 degree Celsius for at least 48 hours before varying concentrations of IPTG from 0 to 500 uM are added. The cells are grown at various temperatures from 30 to 37 degree Celsius for 24 hours before a final GFP reading is taken.

The GFP expression levels correlated well with the temperature to which the cells were subjected to (Figure 1). The highest amount of GFP production were shown at higher temperatures closer to 37 degree Celsius. On the other hand, GFP production is repressed when the temperature is closer to 30 degree Celsius. This is congruent with the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper's] literature (Figure 2 and Figure 4) for the thermal sensitive construct which shows much higher GFP expression at temperatures above 36 degree Celsius.

We have reported an optimum condition of 250 uM of IPTG induction (Figure 3) for our temperature sensitive system. In short, our part could prove useful for any group seeking to elucidate gene expression based on temperature as a physical stimulus.

Figure 1: GFP/OD600nm of thermal sensitive construct after 24 hours incubation at various temperatures.

Figure 2: TlpA36 construct as suggested in the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper.] Our thermal sensitive construct BBa_K2447014 functioned properly as described in this paper; elucidating much higher GFP expression at temperatures higher than 36 degree Celsius.

Figure 3: When 250 uM of IPTG is added, strong GFP expression is elucidated at high temperatures.

Figure 4: Temperature system is activated and shows a one-fold difference in GFP expression between cultures in 30 and 37 degree Celsius, taken after 6 hours.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 1750
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1147
Illegal XhoI site found at 2035
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 2338
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 3303

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2447014 short</partinfo>
-In the absence of IPTG, lacI repressor will bind to pLac promoter and inhibit further downstream expression. pLac promoter is attached upstream of the tlpa36 protein coding sequence to control the amount of tlpa36 protein expression. When IPTG is present and under high temperatures (above 36 degree), pTlpa36 will be constitutively and eliciting downstream GFP expression. Under low temperatures (below 36 degree), the pTlpa36 will be repressed, and drastically reducing GFP expression
+In the absence of IPTG, LacI repressor will bind to pLac promoter and inhibit further downstream expression. pLac promoter is attached upstream of the TlpA36 protein coding sequence to control the amount of TlpA36 protein expression. When IPTG is present and under high temperatures (above 36 degree Celsius), the temperature system will be "ON" and GFP is expressed. Under low temperatures (below 36 degree Celsius), the pTlpA will be repressed, which will stop GFP expression.
 ====Usage and Characterisation====
-This part is inserted into pBbE2k backbone and subsequently characterised in E.coli Beta 10. These cells are grown in LB medium (with kanamycin) at 30 degree for at least 48 hours before varying concentrations of IPTG ions from 0 to 500 uM are added. The cells are grown at various temperatures from 30 to 37 for 24 hours before a final GFP reading is taken.
+For more detailed experimental results, do visit our [http://2017.igem.org/Team:NUS_Singapore/Experiments experimental page].
-The GFP productions correlated well to the temperature in which the cells are subjected to. At higher temperature closer to 37 degree, there was highest amount of GFP production. On the other hand, closer to 30 degree, GFP production is repressed. This is congruent with the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper's]result (figure 2)for the thermal sensitive construct which shows much higher GFP expression at 36 degree and above.
+This part is inserted into pBbE2k backbone and subsequently characterised in <i>E. coli</i> 10beta. These cells are grown in LB medium (with kanamycin) at 30 degree Celsius for at least 48 hours before varying concentrations of IPTG from 0 to 500 uM are added. The cells are grown at various temperatures from 30 to 37 degree Celsius for 24 hours before a final GFP reading is taken.
-At 250uM of iptg, we have reported optimum condition for our temperature sensitive system. In short, our part could prove useful for any group seeking to elucidate gene expression based on temperature as  a physical stimulus.
+The GFP expression levels correlated well with the temperature to which the cells were subjected to (Figure 1). The highest amount of GFP production were shown at higher temperatures closer to 37 degree Celsius. On the other hand, GFP production is repressed when the temperature is closer to 30 degree Celsius. This is congruent with the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper's] literature (Figure 2 and Figure 4) for the thermal sensitive construct which shows much higher GFP expression at temperatures above 36 degree Celsius.
+We have reported an optimum condition of 250 uM of IPTG induction (Figure 3) for our temperature sensitive system. In short, our part could prove useful for any group seeking to elucidate gene expression based on temperature as a physical stimulus.
+[[Image:Temperature1.jpeg|thumb|left|800px| Figure 1: GFP/OD600nm of thermal sensitive construct after 24 hours incubation at various temperatures.]]
+[[Image:Temperature3.png|thumb|right|400px|Figure 2: TlpA36 construct as suggested in the [http://www.nature.com/nchembio/journal/v13/n1/abs/nchembio.2233.html?foxtrotcallback=true paper.] Our thermal sensitive construct BBa_K2447014 functioned properly as described in this paper; elucidating much higher GFP expression at temperatures higher than 36 degree Celsius.]]
+[[Image:Temperature2.png|thumb|center|800px|Figure 3: When 250 uM of IPTG is added, strong GFP expression is elucidated at high temperatures.]]
+[[Image:Temperature4.png|thumb|center|800px|Figure 4: Temperature system is activated and shows a one-fold difference in GFP expression between cultures in 30 and 37 degree Celsius, taken after 6 hours.]]
-[[Image:Temperature1.jpeg|thumb|left|400px|Side by side comparison of construct Bba_K2447000 & BBa_K116404 where GFP/OD is expressed as a percentage. Our proposed part exhibited greater sensitivity to phosphate levels above 40uM]]
-[[Image:Temperature3.png|thumb|right|400px|Our proposed phosphate construct is sensitive to phosphate concentrations from 0 to 1000 uM]]
-[[Image:Temperature2.png|thumb|center|800px|Our proposed phosphate construct is sensitive to phosphate concentrations from 0 to 1000 uM]]
-By replacing the weaker RBS 32 (as utilised by the Taiwanese team) with a stronger binding affinity RBS 34 as proposed by IGEM NUS team 2017, we have elucidated much stronger GFP expression and also improved the sensivity of the part to varying concentrations of phosphate ions. Previously, the Taiwanese part [http://2008.igem.org/Team:NYMU-Taipei/Project/Phosphate had reported]similar level of repressed GFP productions for phosphate concentrations above 40uM. On the other hand, our proposed phosphate sensor is sensitive to phosphate concentration above 40 uM, elucidating differential GFP expressions when placed in different phosphate level.