Difference between revisions of "Part:BBa K2596005"
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<partinfo>BBa_K2596005 parameters</partinfo>
 
<partinfo>BBa_K2596005 parameters</partinfo>
 
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==Sucrose Assays==
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[[File:T--Stony Brook--Experiments moldy WT.jpeg|thumb|Figure 1. Wild Type induced with NaCl and possibly contaminated with mold]]
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[[File:T--Stony Brook--Experiments moldy.jpeg|thumb|Figure 2 Cyanobacterial culture contaminated by unknown species of fungus]]
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In order to test CscB (sucrose permease), we added 150 mM of NaCl to 250 mL erlenmeyer flasks with 50 mL of BG-11 to induce sugar production. S. elongatus PCC 7942 naturally produces sucrose to balance out the osmotic pressure caused by the extracellular salt. Additionally, we added 2 g/L of pH 8.0 HEPES buffer to prevent acidification of the media (as cscB depends on a basic environment to function). We also induced with 1 mM of IPTG after the cells had reached the mid-log phase.
 +
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The Sucrose/D-glucose assay kit was donated to us by Megazyme and was tested on the cyanobacteria after 1 week and then 2 weeks of IPTG induction. The cyanobacteria were spinned down at 10,000 g for ~5-10 minutes until a pellet formed, and 200 µL of their supernatant was extracted for the assay.
 +
With the assay, the sample was mixed with both acetate buffer (negative control) and fructosidase. After incubating at 50 ℃ for 20 minutes, we added the GOPOD dye, which turns a bright pink (510 nm) when exposed to glucose. After incubating for the required time, we ran triplicates in a spectrophotometer and calculated glucose values with the calculator on Megazyme’s website.
 +
 +
We tested all of our samples against glucose and starch controls (0.25g/L of flour), as well as uninduced samples of cyanobacteria (which lacked NaCl and/or IPTG).
 +
 +
After the insignificant results, we measured the pH because cscB will not secrete sucrose in acidic conditions. Before even testing for sucrose, we noticed that the wild type culture (without antibiotics) had gotten visibly contaminated by fungus (figure 2). As such, we suspect contamination to be the major cause of our lack of results:
 +
 +
Then, we started a new round of testing, with and without added antibiotics. We conducted the sucrose assay as before, except the cultures with antibiotics were supplied with 10 µL spectinomycin and 10 µL streptomycin. The cultures we measured sucrose for are:
 +
#1 = cscB #3 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 25 ℃
 +
#2 = cscB #2 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 25 ℃
 +
#3 = cscB #2 0 mM NaCl 1 mM IPTG 33 ℃
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#4 = cscB #2 150 mM NaCl 1 mM IPTG 33 ℃
 +
#5 = cscB #2 150 mM NaCl 33 ℃
 +
#6 = cscB #4 #1 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
 +
#7 = cscB #4 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
 +
#8 = cscB #2 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
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#9 = cscB #4  #2 150 mM NaCl 1 mM IPTG 33 ℃

Revision as of 16:50, 16 October 2018


cscB (sucrose permease) from E. coli

This is a sucrose symporter protein from E. Coli.

Usage and Biology

CscB is a cell membrane protein that plays an integral role in transporting sucrose across the membrane as it is a sucrose-proton symporter. It is compatible in many strains of S. elongatus, including 2973, 6803 and 7942. S. elongatus is unique in that its cell membrane proton gradient is generated by maintaining a surplus of protons inside the cell. Sucrose can then be exported from the cell using the proton gradient. 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 481
    Illegal NgoMIV site found at 604
  • 1000
    COMPATIBLE WITH RFC[1000]


Sucrose Assays

Figure 1. Wild Type induced with NaCl and possibly contaminated with mold
Figure 2 Cyanobacterial culture contaminated by unknown species of fungus

In order to test CscB (sucrose permease), we added 150 mM of NaCl to 250 mL erlenmeyer flasks with 50 mL of BG-11 to induce sugar production. S. elongatus PCC 7942 naturally produces sucrose to balance out the osmotic pressure caused by the extracellular salt. Additionally, we added 2 g/L of pH 8.0 HEPES buffer to prevent acidification of the media (as cscB depends on a basic environment to function). We also induced with 1 mM of IPTG after the cells had reached the mid-log phase.

The Sucrose/D-glucose assay kit was donated to us by Megazyme and was tested on the cyanobacteria after 1 week and then 2 weeks of IPTG induction. The cyanobacteria were spinned down at 10,000 g for ~5-10 minutes until a pellet formed, and 200 µL of their supernatant was extracted for the assay. With the assay, the sample was mixed with both acetate buffer (negative control) and fructosidase. After incubating at 50 ℃ for 20 minutes, we added the GOPOD dye, which turns a bright pink (510 nm) when exposed to glucose. After incubating for the required time, we ran triplicates in a spectrophotometer and calculated glucose values with the calculator on Megazyme’s website.

We tested all of our samples against glucose and starch controls (0.25g/L of flour), as well as uninduced samples of cyanobacteria (which lacked NaCl and/or IPTG).

After the insignificant results, we measured the pH because cscB will not secrete sucrose in acidic conditions. Before even testing for sucrose, we noticed that the wild type culture (without antibiotics) had gotten visibly contaminated by fungus (figure 2). As such, we suspect contamination to be the major cause of our lack of results:

Then, we started a new round of testing, with and without added antibiotics. We conducted the sucrose assay as before, except the cultures with antibiotics were supplied with 10 µL spectinomycin and 10 µL streptomycin. The cultures we measured sucrose for are:

  1. 1 = cscB #3 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 25 ℃
  2. 2 = cscB #2 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 25 ℃
  3. 3 = cscB #2 0 mM NaCl 1 mM IPTG 33 ℃
  4. 4 = cscB #2 150 mM NaCl 1 mM IPTG 33 ℃
  5. 5 = cscB #2 150 mM NaCl 33 ℃
  6. 6 = cscB #4 #1 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
  7. 7 = cscB #4 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
  8. 8 = cscB #2 150 mM NaCl 1 mM IPTG 10 uM Spec 10 uM Strep 33 ℃
  9. 9 = cscB #4 #2 150 mM NaCl 1 mM IPTG 33 ℃