Difference between revisions of "Part:BBa K2235007"

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===Important parameters===
 
===Important parameters===
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Table 1: Parameters for OmpR sialidase biobrick (BBa_K2235007)
 
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===Expression and purification of sialidase===
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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 1-3. 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.
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Picture
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Picture
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Picture
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===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. 
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[[File:BBa_M30011 osmograph11.png|600px|thumb|left|Figure 3]]

Revision as of 13:17, 18 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
Tag C-terminal 6xHis
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 1-3. 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.

Picture

Picture

Picture

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 3