Difference between revisions of "Part:BBa K2235007"

Latest revision as of 16:11, 27 October 2017

OmpR promoter regulating the expression of sialidase enzyme

OmpR is an osmotic pressure promoter. It can sense changes in for example sucrose concentration. A higher concentration of sucrose will lead to a higher expression of the gene after the OmpR promoter. In this biobrick the gene after OmpR is an enzyme called sialidase. This enzyme cuts sialic acid (that's for example found in mucus). The sialidase produced by this biobrick has a HIS-tag on it to make it easy to purify.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 189
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 636
Illegal NgoMIV site found at 711
Illegal NgoMIV site found at 801
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1181

Usage and biology

OmpR is an already well characterized biobrick (BBa_R0082). It is a promoter that takes advantage of the OmpR/EnvZ two-component system, which is utilized by many bacteria, to sense the change in osmotic pressure. We connected this promoter to the coding sequence for the enzyme sialidase. This enzyme hydrolyzes glycosidic linkages of terminal sialic acid residues in glycoproteins. This composite biobrick can be used to produce sialidase when the osmotic pressure in the environment around the bacteria increases.

Important parameters

Table 1: Parameters for OmpR sialidase biobrick (BBa_K2235007)

Experiment	Characteristic	Result
Expression
	Compatibility	E. coli TOP10 E. coli ΔEnvZ
	Promoter	OmpR
	Optimal temperature	37 °C
	Number of amino acids	XXX
	Molecular weight	XXXnbsp;kDa
Purification
Purification	Tag	C-terminal 6xHis
Sialidase expression
Sialidase expression	SDS-PAGE	Sialidase expression seen in some tests
Osmotic pressure detection
	Sucrose	Works
	NaCl	Non-conclusive data

Expression and purification of sialidase

We tested our biobrick by cultivating transformed bacteria (both TOP10 and ΔEnvZ) in different concentrations of sucrose, 0, 5, 10 and 15%. After a desired OD was reached (we used OD 0.4) we sonicated the cells to allow for purification of sialidase. The sialidase is connected to a His-tag so we used IMAC columns to purify the enzyme. The purified enzymes were then analyzed by SDS-PAGE, see figure 1. The figures shows some vague bands of sialidase for some of the concentrations, however this result is not conclusive enough to say anything about the gradient dependent expression. It is possible to say that the biobrick can produce sialidase, but more test would need to be done and we did not have enough time to finish them.

Figure 1

Figure 1. SDS-PAGE gel of sialidase expression. From left to right: ladder, well 2-3: expression in 5% sucrose, well 4-8: expression in 10% sucrose, well 9-14: expression in 15% sucrose, well 15: positive control.

Osmotic pressure detection

Before we ligated the OmpR promoter to sialidase we tested how the OmpR promoter works. We found an already existing biobrick for this (BBa_M30011) that had OmpR connected to RFP as a reporter. We designed an osmotic pressure test by the help of iGEM 2015 who already had worked with this biobrick. In the test we cultivated TOP10 cells in different sucrose and NaCl concentrations and measured the fluorescence of RFP at different OD points (for more detailed explanation see the experience page for BBa_M30011). The results we got showed that OmpR produced more RFP as the osmotic pressure increased by sucrose, see figure 4-5. However for NaCl we saw no correlation between the increase in osmotic pressure and RFP expression, see figure 6-7. We therefore decided to only use sucrose when we tested our final biobrick.

Figure 2

Figure 2: Expression of RFP fluorescence after activation of the osmotic pressure sensitive promoter OmpR with different sucrose concentrations. As the sucrose concentration increases, and by so the osmotic pressure rises, the RFP expression is increased.

Figure 3

Figure 3: Expression of RFP fluorescence plotted against the OD-value at which the sample was taken. The higher sucrose concentrations did not reach the highest OD-values and are therefore missing some points.

Figure 4

Figure 4: Expression of RFP fluorescence after activation of the osmotic pressure sensitive promoter OmpR with different NaCl concentrations. No correlation can be seen from this graph.

Figure 5

Figure 5: Expression of RFP fluorescence plotted against the OD-value at which the sample was taken. No correlation can be seen from this graph.

Conclusion

What we have shown with these experiments is that BBa_K2235007 can produce sialidase. Its promoter should be able to detect a change in osmotic pressure due to increase in sucrose concentration, but our test gave no results for that. Since the test for sialidase expression was performed the last weeks before the Giant Jamboree we did not have time to redo the test. We think that if the test were performed again we would see the same increase of sialidase as we saw for RFP.

@@ Line 22: / Line 22: @@
 ===Important parameters===
-<center>
+Table 1: Parameters for OmpR sialidase biobrick (BBa_K2235007)
 {|{{Table}}
 !Experiment
@@ Line 28: / Line 28: @@
 !Result
 |-
-|rowspan="5"|Expression
+|rowspan="6"|Expression
 |-
 |Compatibility
@@ Line 44: / Line 44: @@
 |-
 |Molecular weight
-|XXX;kDa
+|XXXnbsp;kDa
 |-
-|rowspan="6"|Purification
+|rowspan="2"|Purification
-|-
-|Extinction coefficient at 280 nm <br> measured in water [M<sup>-1</sup> cm<sup>-1</sup>]
-|37400 (assuming all pairs of Cys residues form cystines)
-(assuming all Cys residues are reduced)
 |-
 |Tag
 |C-terminal 6xHis
 |-
-|rowspan="2"|NAD<sup>+</sup> detection
+|rowspan="2"|Sialidase expression
+|-
+|SDS-PAGE
+|Sialidase expression seen in some tests
+|-
+|rowspan="3"|Osmotic pressure detection
 |-
-|Limit of detection
+|Sucrose
-|2 nM
+|Works
 |-
+|NaCl
+|Non-conclusive data
 |}
-</center>
+===Expression and purification of sialidase===
+We tested our biobrick by cultivating transformed bacteria (both TOP10 and ΔEnvZ) in different concentrations of sucrose, 0, 5, 10 and 15%. After a desired OD was reached (we used OD 0.4) we sonicated the cells to allow for purification of sialidase. The sialidase is connected to a His-tag so we used IMAC columns to purify the enzyme. The purified enzymes were then analyzed by SDS-PAGE, see figure 1. The figures shows some vague bands of sialidase for some of the concentrations, however this result is not conclusive enough to say anything about the gradient dependent expression. It is possible to say that the biobrick can produce sialidase, but more test would need to be done and we did not have enough time to finish them.
+[[File:OmpR sia SDS.png|600px|thumb|left|Figure 1]]
+Figure 1. SDS-PAGE gel of sialidase expression. From left to right: ladder, well 2-3: expression in 5% sucrose, well 4-8: expression in 10% sucrose, well 9-14: expression in 15% sucrose, well 15: positive control.
+===Osmotic pressure detection===
+Before we ligated the OmpR promoter to sialidase we tested how the OmpR promoter works. We found an already existing biobrick for this (BBa_M30011) that had OmpR connected to RFP as a reporter. We designed an osmotic pressure test by the help of iGEM 2015 who already had worked with this biobrick. In the test we cultivated TOP10 cells in different sucrose and NaCl concentrations and measured the fluorescence of RFP at different OD points (for more detailed explanation see the experience page for BBa_M30011). The results we got showed that OmpR produced more RFP as the osmotic pressure increased by sucrose, see figure 4-5. However for NaCl we saw no correlation between the increase in osmotic pressure and RFP expression, see figure 6-7. We therefore decided to only use sucrose when we tested our final biobrick.
+[[File:Osmo RFP.png|600px|thumb|left|Figure 2]]
+Figure 2: Expression of RFP fluorescence after activation of the osmotic pressure sensitive promoter OmpR with different sucrose concentrations. As the sucrose concentration increases, and by so the osmotic pressure rises, the RFP expression is increased.
+[[File:Osmo OD RFP.png|600px|thumb|left|Figure 3]]
+Figure 3: Expression of RFP fluorescence plotted against the OD-value at which the sample was taken. The higher sucrose concentrations did not reach the highest OD-values and are therefore missing some points.
+[[File:Osmo NaCl RFP.png|600px|thumb|left|Figure 4]]
+Figure 4: Expression of RFP fluorescence after activation of the osmotic pressure sensitive promoter OmpR with different NaCl concentrations. No correlation can be seen from this graph.
+[[File:Osmo NaCl OD RFP.png|600px|thumb|left|Figure 5]]
+Figure 5: Expression of RFP fluorescence plotted against the OD-value at which the sample was taken. No correlation can be seen from this graph.
+===Conclusion===
+What we have shown with these experiments is that BBa_K2235007 can produce sialidase. Its promoter should be able to detect a change in osmotic pressure due to increase in sucrose concentration, but our test gave no results for that. Since the test for sialidase expression was performed the last weeks before the Giant Jamboree we did not have time to redo the test. We think that if the test were performed again we would see the same increase of sialidase as we saw for RFP.