Difference between revisions of "Part:BBa K584028"

Latest revision as of 15:41, 1 October 2011

pLac-Lux hybrid promoter + INP(Ice Nucleating Protein Generator)

The pLac-Lux hybrid promoter is repressed by the lacI repressor and induced by IPTG. It is repressed by the luxR repressor bound to the corepressor CO6HSL. It contains a lux box and a lacI binding site. The "Design Notes" portion of this page contains a truth table for this logic gate.

The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria. inaZ is the gene responsible for producing the ice nucleation protein.

We would like to thank [http://www.academicbiography.uwo.ca/profile.php?&n=ggloor Prof. Dr. Gregory Gloor ], for providing us with a plasmid (pUC1813ICE) which contains the ice nucleating protein gene inaZ.

To know more about the inaZ, it is suggested to read this [http://www.ncbi.nlm.nih.gov/pubmed/10933828 article] describing the usage of pUC1813ICE with the inaZ.

Characterization

Note: to generate supercooled water, ultrapure “milli-Q” water (Millipore Corporation) was filtered over a 0.22µm filter to generate water devoid of nucleating species. All experiments were performed in TOP10F' cells.

This experiment can be conducted at temperatures between -4°C and -10°C. At -10°C the water automatically freezes in the alcohol bath. This test has been done multiple times at -6°C in a reproductive way. At -4°C the freezing of water is unpredictable; sometimes it freezes and sometimes it doesn't. So we assume that -10°C and -4°C are the limits for the ice nucleation by a lactose stimulated E.D. Frosti. Further characterization can be done when smaller temperature intervals are being used. Unfortunately, we did not have the time to further characterize BBa_K584028 at this moment.

An overnight preculture was inoculated at an initial OD of 0.1 in 15mL medium without or with (1mM) IPTG, and grown for 5 hours at 37°C. Cells were spun down and resuspended in 1 mL filtered water, after which they were washed twice with 1mL filtered water. The ODs of the resulting cell suspensions were determined and all cells were diluted to the same OD of 10.
In the meanwhile, the ethanol bath was set at -6°C, and clean, plastic tubes filled with 5mL filtered water were put in the chilled bath to create supercooled water at -6°C.

For the INP test, the same amount of induced (with IPTG)/non-induced (no IPTG) cells were added to the 5mL supercooled water and checked for ice nucleating activity (see videos).

As can be seen on the videos, addition of cells induced to express INP (1mM IPTG) clearly induced ice crystallization. However, also the cells which were grown without the addition of IPTG displayed ice nucleating activity. It thus seems that the pLac-lux promoter is leaky to an extent which results in ice-nucleating activity under our experimental conditions. This leakiness in TOP10F’ was not detected using a GFP reporter assay system (see characterization of our biobrick BBa_K584002). The ice nucleating activity therefore appears to be a more sensitive reporter system to test for promoter activity, which is in agreement with literature indicating the sensitivity of INP as a reporter for promoter analysis (1,2).

References

1. Drainas C, Vartholomatos G, Panopoulos NJ: The Ice Nucleation Gene from Pseudomonas syringae as a Sensitive Gene Reporter for Promoter Analysis in Zymomonas mobilis. Appl Environ Microbiol 1995, 61(1):273-277.

2. Lindgren PB, Frederick R, Govindarajan AG, Panopoulos NJ, Staskawicz BJ, Lindow SE: An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola. EMBO J 1989, 8(5):1291-1301.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 746
Illegal NgoMIV site found at 2258
Illegal NgoMIV site found at 2402
Illegal NgoMIV site found at 2666
Illegal NgoMIV site found at 2810
Illegal NgoMIV site found at 2858
Illegal NgoMIV site found at 2978
Illegal AgeI site found at 517
Illegal AgeI site found at 1922
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 692
Illegal BsaI.rc site found at 3438
Illegal SapI.rc site found at 338

@@ Line 9: / Line 9: @@
 <p align="justify">To know more about the inaZ, it is suggested to read this [http://www.ncbi.nlm.nih.gov/pubmed/10933828 article] describing the usage of pUC1813ICE with the '''inaZ'''.</p>
+== '''Characterization''' ==
+''Note'': to generate supercooled water, ultrapure “milli-Q” water (Millipore Corporation) was filtered over a 0.22µm filter to generate water devoid of nucleating species. All experiments were performed in TOP10F' cells. <br>
+<b>This experiment can be conducted at temperatures between -4°C and -10°C. At -10°C the water automatically freezes in the alcohol bath. This test has been done multiple times at -6°C in a reproductive way. At -4°C the freezing of water is unpredictable; sometimes it freezes and sometimes it doesn't. So we assume that -10°C and -4°C are the limits for the ice nucleation by a lactose stimulated <i>E.D. Frosti</i>. Further characterization can be done when smaller temperature intervals are being used. Unfortunately, we did not have the time to further characterize <i>BBa_K584028</i> at this moment.</b><br>
+An overnight preculture was inoculated at an initial OD of 0.1 in 15mL medium without or with (1mM) IPTG, and grown for 5 hours at 37°C. Cells were spun down and resuspended in 1 mL filtered water, after which they were washed twice with 1mL filtered water. The ODs of the resulting cell suspensions were determined and all cells were diluted to the same OD of 10.<br>
+In the meanwhile, the ethanol bath was set at -6°C, and clean, plastic tubes filled with 5mL filtered water were put in the chilled bath to create supercooled water at -6°C. <br><br>
+<b>For the INP test, the same amount of induced (with IPTG)/non-induced (no IPTG) cells were added to the 5mL supercooled water and checked for ice nucleating activity (see videos).</b><br><br>
+<html>
+<center>
+<iframe width="560" height="315" src="http://www.youtube.com/embed/Pb958DzMkuc" frameborder="0" allowfullscreen></iframe><br><br>
+<iframe width="560" height="315" src="http://www.youtube.com/embed/TnjoLeih6QE" frameborder="0" allowfullscreen></iframe><br><br>
+</center>
+</html>
+<br><br>
+As can be seen on the videos, addition of cells induced to express INP (1mM IPTG) clearly induced ice crystallization. However, also the cells which were grown without the addition of IPTG displayed ice nucleating activity. It thus seems that the pLac-lux promoter is leaky to an extent  which results in ice-nucleating activity under our experimental conditions. This leakiness in TOP10F’ was not detected using a GFP reporter assay system (see characterization of our biobrick [https://parts.igem.org/wiki/index.php?title=Part:BBa_K584002 BBa_K584002]).
+The ice nucleating activity therefore appears to be a more sensitive reporter system to test for promoter activity, which is in agreement with literature indicating the sensitivity of INP as a reporter for promoter analysis (1,2).<br><br>
+== '''References''' ==
+.	Drainas C, Vartholomatos G, Panopoulos NJ: The Ice Nucleation Gene from Pseudomonas syringae as a Sensitive Gene Reporter for Promoter Analysis in Zymomonas mobilis. Appl Environ Microbiol 1995, 61(1):273-277.<br><br>
+.	Lindgren PB, Frederick R, Govindarajan AG, Panopoulos NJ, Staskawicz BJ, Lindow SE: An ice nucleation reporter gene system: identification of inducible pathogenicity genes in Pseudomonas syringae pv. phaseolicola. EMBO J 1989, 8(5):1291-1301.