Difference between revisions of "Team:Exeter/Parts"

Revision as of 14:44, 18 October 2016

Parts

KillerRed

Name

pT7- E. coli optimised - KillerRed (EOKR)

Description

KillerRed is a red fluorescent protein that generates reactive oxygen species after illumination with light between the wavelengths of 540-580 nm[1].

We further characterised this kill switch by illuminating induced cultures 24 hours after induction with IPTG as well as uninduced cultures. After 6 hours in the light box CFU’s were counted to determine if the kill switch was successful. This was also reproduced on cultures grown in a ministat for 120 and 168 hours to test how long the kill switch remains functional. The full protocol can be found here and the results can be found here.

The mechanism by which KillerRed kills cells isn’t fully understood yet

Here we are submitting KillerRed as a composite part under a T7 promoter (BBa_I712074), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).

The sequence for KillerRed protein coding region can be found here (BBa_K1141002)

Biobrick Code

BBa_K1914003

KillerOrange

Name:

pT7- E. coli optimised - KillerOrange (EOKO)

Description:

KillerOrange is a mutant of the fluorescent protein KillerRed (BBa_K1141002, BBa_K1491015) activated by blue and green light. It carries a tryptophan-based chromophore that is novel for photosensitizers [2].

KillerOrange has an excitation maximum of 512 nm and emission maximum at 555 nm and its absorbance spectrum has two peaks, at 455 and 514 nm.

The mechanism by which KillerOrange kills cells isn’t fully understood yet. However, it is believed that KillerOrange's ability to generate reactive oxygen species depends on a water-filler channel reaching the chromophore area from the end cap of the ß-barrel [3].

We characterised this part in the same way as KillerRed (protocol), and the results of which can be found here

Here we are submitting KillerOrange as a composite part under a T7 promoter (BBa_I712074), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).

The sequence for KillerOrange protein coding region can be found here (BBa_K1914000)

Biobrick Code

BBa_K1914001

Lysozyme

Name:

pT7 Lysozyme E. coli codon optimised, signal peptide, flag tag

Description:

Lysozyme from chicken egg white, attacks the cell wall in bacteria by hydrolysing the ß-1,4 linkages between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan [4].

Characterisation of this part involved using the Enzcheck assay kit which detects lysozyme activity as a measure of fluorescence as well as using the same protocol we used to characterise KillerRed (protocol)and the results of which can be found here

Here we are submitting lysozyme as a composite part under a T7 promoter (BBa_I712074), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).

We have codon optimised the protein coding region, added a FLAG tag and exchanged the native signal peptide for one which is E. coli specific and directs Lysozyme to the periplasm. The sequence for Lysozyme protein coding region can be found here (BBa_K1914004)

Biobrick Code

BBa_K1914005

DNase

Name:

DNase

Description:

DNase was designed as a DNA degrading kill switch aiming to prevent cross contamination of DNA

This part was not successfully transformed. This could be due to the need for a tighly regulating promoter to prevent DNase protein being produced before induction and destroying DNA.

References

Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., Kotera, I., Arai, Y., Horiuchi, M., Fukui, K. and Ayabe, T., 2013. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Scientific reports, 3.
Sarkisyan, K.S., Zlobovskaya, O.A., Gorbachev, D.A., Bozhanova, N.G., Sharonov, G.V., Staroverov, D.B., Egorov, E.S., Ryabova, A.V., Solntsev, K.M., Mishin, A.S. and Lukyanov, K.A., 2015. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PloS one,10(12), p.e0145287.
Pletnev, S., Gurskaya, N.G., Pletneva, N.V., Lukyanov, K.A., Chudakov, D.M., Martynov, V.I., Popov, V.O., Kovalchuk, M.V., Wlodawer, A., Dauter, Z. and Pletnev, V., 2009. Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. Journal of Biological Chemistry, 284(46), pp.32028-32039.
Blake, C.C.F., Koenig, D.F., Mair, G.A., North, A.C.T., Phillips, D.C. and Sarma, V.R., 1965. Structure of hen egg-white lysozyme: a three-dimensional Fourier synthesis at 2 Å resolution. Nature, 206(4986), pp.757-761.

@@ Line 655: / Line 655: @@
                  <h6>Name</h6>
-                 <p id="pp">pT7- E. coli optimised - KillerRed (EOKR)</p>
+                 <p id="pp">pT7- <i>E. coli</i> optimised - KillerRed (EOKR)</p>
                  <h6>Description</h6>
@@ Line 690: / Line 690: @@
 <h6>Name:</h6>
-                 <p id="pp">pT7- E. coli optimised - KillerOrange (EOKO)</p>
+                 <p id="pp">pT7- <i>E. coli</i> optimised - KillerOrange (EOKO)</p>
                  <h6>Description:</h6>
@@ Line 729: / Line 729: @@
 <h6>Name:</h6>
-                 <p id="pp">pT7 Lysozyme  E.coli codon optimised, signal peptide, flag tag</p>
+                 <p id="pp">pT7 Lysozyme  <i>E. coli</i> codon optimised, signal peptide, flag tag</p>
                  <h6>Description:</h6>
@@ Line 740: / Line 740: @@
                  <p id="pp">Here we are submitting lysozyme as a composite part under a T7 promoter (BBa_I712074), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).</p>
-                 <p id="pp">We have codon optimised the protein coding region, added a FLAG tag and exchanged the native signal peptide for one which is E. coli specific and directs Lysozyme to the periplasm. The sequence for Lysozyme protein coding region can be found here (BBa_K1914004)</p>
+                 <p id="pp">We have codon optimised the protein coding region, added a FLAG tag and exchanged the native signal peptide for one which is <i>E. coli</i> specific and directs Lysozyme to the periplasm. The sequence for Lysozyme protein coding region can be found here (BBa_K1914004)</p>
                  <h6> Biobrick Code </h6>