Team:Cornell/testing/project/wetlab/4/4

Western Blots for MtrB

To assess translational efficiency of mtrB in our constructs, we will be conducting western blots for MtrB. Because we were unable to obtain MtrB antibodies, we decided to use the commonly used cell biological method of appending poly-His tags for detection by poly-His antibodies. We are in the process of separately adding poly His tags with short Gly linkers to the N and C termini of p14K, p16K, SAL, and SAL2 for a total of 8 potential biobricks. In addition, the attachment of these His tags will facilitate purification of MtrB which may have future application for in vitro characterization of MtrB. The primers used were as follows:

p14K

A. Poly-His Tag at N-terminus
1. F: 5'- CATCACCAT-GGATCC-ATG- CATCACCATCACCATCAC-GGAAGTGGA- AAATTTAAACTGAATTTG- 3' (Dummy sequence- BamHI- Start codon- His Tag- Linker- mtrB)
2. R: REV_PRIME_mtrB
B. Poly-His Tag at C-terminus
3. F: FOR_Prime_mtrB
4. R: 5'- CATCACCAT-GGTACC- TTA-GTGATGGTGATGGTGATG-TCCACTTCC-GAGTTTGTAACTCATGCT- 3' (Dummy Sequence- KpnI- Stop Codon- His Tag- Linker- mtrB)

p16K

C. Poly-His Tag at N-terminus
F: 5'- CATCACCAT- GGCGCGCC- ATG- CATCACCATCACCATCAC-GGAAGTGGA- AAATTTAAACTGAATTTG- 3' (Dummy Sequence- AscI- Start Codon- His Tag-Linker- mtrB)
R: 5' - CATCACCAT- ACTAGT- TTAGAGTTTGTAACTCATGCT- 3' (Dummy Sequence- SpeI- mtrB)
D. Poly-His at C-terminus
F: 5'- CATCACCAT- GGCGCGCC- ATGAAATTTAAACTGAATTTG- 3' (Dummy Sequence- AscI- mtrB)
R: 5' - CATCACCAT- ACTAGT- TTA- GTGATGGTGATGGTGATG-TCCACTTCC-GAGTTTGTAACTCATGCT- 3' (Dummy Sequence- SpeI- Stop Codon- His Tag- Linker- mtrB)

Sal 1

Same as A and B

Sal 2

Same as C and D