Construction of pUC19 Nag1/Uap1 expression construct and characterization via protein expression in E.coli

Enzymatic Digestions

Digested 2 ug pUC19 with Xba1. 2 reactions, 2 ul (1ug) of plasmid with 2 ul Xba1, 2.5ul Buffer 4, 2.5 ul 10x BSA, 18ul dH20. Digested for 1 hour at 37deg. Heat inactivated for 20 minutes at 70deg. Ran through PCR cleanup column. Eluted with 50 ul TE buffer.
Digested nag1 and Uap1 inserts with Xba1 and Spe1. Used 18 ul of insert (low concentration) with 1 ul each enzyme, 2.5 ul buffer and 2.5ul 10x bsa. Digested for 1 hour, heat inactivated for 20 minutes at 70C. ran through clean up columns and eluted with 50 ul TE buffer.

Gel Quantitation

Phosphatase treatment and Ligations

Treated digested puc19 with Antarctic phosphatase (NEB).
50ul of purified plasmid = ~20ug/ul
Used 9ul digested and purified plasmid /1 ul buffer/1ul phosphatase (10ul total reaction). Incubated for 15 minutes at 37degC and heat inactivated at 70 deg C for 5min.
Two different ligation reactions were performed, either 1ul or 2 ul of AP treated backbone was used, for either 17ul or 16ul of insert, respectively + 2ul buffer and 1 ul of T4 ligase (NEB) (20ul total reaction)
Incubated at room temp for 15 minutes.

Transformations

5 ul of reaction was used per transformation using JM109 E. coli made competent using the Transforaid kit from thermo scientific. 50 ul of cells were plated onto prewarmed LB-Amp plates and grown overnight.
Successful transformants of JM109. Negative control is phosphatase treated backbone alone with no insert added during ligations.
Since insert may be in either orientation (Xba1 and Spe1 have complementary sticky ended overhangs) Colonies will be tested for proper insertion via digestion.

Protein expression

The Novagen BugBuster Master Mix protein extraction reagent was used to extract total cell protein from transformed E. coli.
Cells were grown overnight in 5 mls of LB-AMP liquid media along with 0.5 mM IPTG for induction. Cells were then spun down at 4300 RPM at 4deg C for 15min.
Cell pellet was frozen and thawed at -20degC, resuspended in 1ml of BugBuster Master Mix and transferred into 1.5ml microfuge tubes which were then slowly shaken at room temperature on an orbital platform set to 90 rpm.
Lysed samples were then centrifuged for 20min at 4deg C.
25ul of supernatant was resuspended in an equal volume of 2X Laemmeli loading buffer containing beta-mercaptoethanol and heated to 100 deg C for 5 minutes.
20 ul of total denatured protein extracts of cells overexpressing either Nag1 or Uap1 protein were then loaded into a 12% polyacrylamide gel and run at 200 volts for 40 minutes.


Gels were then stained with Coomasie dye for 3 hours and then de-stained.

As can be seen from the gel data, it appears that the Nag1 protein is being overexpressed in approximately half of the transformed strains (lanes 4-8), as expected. The expected size is 27.5 kD. The Uap1 protein however does not appear to be overexpressed in the transformed strains. The band corresponding to the putative Nag1 protein has been excised from the gel and will be subjected to mass spectrometry for further confirmation.