Difference between revisions of "Part:BBa K2447014"

Revision as of 07:54, 25 October 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 degrees), pTlpA36 will be "ON" expressing GFP. Under low temperatures (below 36 degrees), the pTlpA36 will be repressed, which will stop GFP expression.

Usage and Characterisation

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 degrees 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 degrees for 24 hours before a final GFP reading is taken.

The GFP expression levels correlated well to the temperature to which the cells were subjected to (Figure 1). At higher temperature closer to 37 degrees, there was highest amount of GFP production. On the other hand, closer to 30 degrees, 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 and Figure 4) for the thermal sensitive construct which shows much higher GFP expression at 36 degrees and above.

At 250uM of IPTG (Figure 3), 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.

Figure 1: GFP/OD 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 had functioned properly as described in this paper; elucidating much higher GFP expression at temperatures higher than 36 degrees.

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

Figure 4: Temperature system is activated and is showing one-fold difference in GFP expression between cultures in 30 and 37 degrees.

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 degrees), pTlpA36 will be "ON" expressing GFP. Under low temperatures (below 36 degrees), the pTlpA36 will be repressed, which will stop 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 degrees), pTlpA36 will be "ON" expressing GFP. Under low temperatures (below 36 degrees), the pTlpA36 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.
+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 degrees 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 degrees for 24 hours before a final GFP reading is taken.
-The GFP productions correlated well to the temperature in which the cells are subjected to (figure 1). 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 and figure 4) for the thermal sensitive construct which shows much higher GFP expression at 36 degree and above.
+The GFP expression levels correlated well to the temperature to which the cells were subjected to (Figure 1). At higher temperature closer to 37 degrees, there was highest amount of GFP production. On the other hand, closer to 30 degrees, 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 and Figure 4) for the thermal sensitive construct which shows much higher GFP expression at 36 degrees and above.
-At 250uM of IPTG (figure 3), 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.
+At 250uM of IPTG (Figure 3), 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.
 [[Image:Temperature1.jpeg|thumb|left|800px| Figure 1: GFP/OD 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 had functioned properly as described in this paper; elucidating much higher GFP expression at temperatures higher than 36 degree.]]
+[[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 had functioned properly as described in this paper; elucidating much higher GFP expression at temperatures higher than 36 degrees.]]
 [[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 indicated by the one-fold increase in GFP expression when the temperature is at 37 degree. At 30 degree, tlpa36 proteins de-dimerises and attach to the promoter tlpa36, and repress it,thereby reducing downstream expression of GFP.]]
+[[Image:Temperature4.png|thumb|center|800px|Figure 4: Temperature system is activated and is showing one-fold difference in GFP expression between cultures in 30 and 37 degrees.]]