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Team:BYU Provo/Notebook/Cholera - Enzyme/October/Period2/Dailylog
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We purified both our AmyA and DspB protein products using the His-tag purification protocol listed on the protocols page. The protein products were then run on a protein gel and [put results] | We purified both our AmyA and DspB protein products using the His-tag purification protocol listed on the protocols page. The protein products were then run on a protein gel and [put results] | ||
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| + | <font size="4">'''10/23/13'''</font> | ||
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| + | We started growing up cholera overnights to run our biofilm degredation assay with our newly purified DspB and AmyA. Samples 1-24 were treated at T = 0 to test for enzymatic inhibition of biofilm growth. Samples 25-48 were treated at T = 48 to test for enzymatic degradation of the biofilm. Treatments were administered according to the following: | ||
| + | Biofilm Degradation Overnights | ||
| + | 1-3 Control | ||
| + | 4-6 Urea | ||
| + | 7-9 2.5 uL DspB | ||
| + | 10-12 12.5 uL DspB | ||
| + | 13-15 25 uL DspB | ||
| + | 16-18 2.5 uL AmyA | ||
| + | 19-21 12.5 uL AmyA | ||
| + | 22-24 25 uL AmyA | ||
| + | 25-27 Control | ||
| + | 28-30 Urea | ||
| + | 31-33 2.5 uL DspB | ||
| + | 34-36 12.5 uL DspB | ||
| + | 37-39 25 uL DspB | ||
| + | 40-42 2.5 uL AmyA | ||
| + | 43-45 12.5 uL AmyA | ||
| + | 46-48 25 uL AmyA | ||
<br> | <br> | ||
Revision as of 23:28, 28 October 2013
| Cholera - Enzymes Notebook: October 15 - October 31 Daily Log
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10/15/13 We ran a gel for the colony PCR products we started last night and colony 5 showed a bright band right around 1100 bp's which is the right size for DspB. We then started a 5 ml overnight in LB-Amp from its corresponding streak on the plate.
10/16/13 We ran plasmid purification on our DspB Colony E from the O/N we started yesterday. We then transformed the plasmid into BL21, which is the E. coli strain that we use for expression and purification. The transformation product was plated on LB+AMP and left in the 37° incubator overnight. All procedures were performed according to the protocols listed on our protocols page.
10/17/13 We started a 5 mL overnight of our DspB in BL21 from the plate on 10/16. We also added the AMP to our autoclaved LB. Now we just have to finish waiting for our BL21 to grow up.
10/18/13 We inoculated 500 mL LB+AMP with the 5 mL overnight from yesterday and allowed it to grow for three hours from 4 am to 7 am. At 7 am we added 500 uL IPTG to induce expression of our protein and continued to grow it for another five hours from 7 am to 12 pm. At 12 pm we pelleted the bacteria and placed it in the -80°. We will purify this protein product on Monday.
10/21/13 We purified both our AmyA and DspB protein products using the His-tag purification protocol listed on the protocols page. The protein products were then run on a protein gel and [put results]
10/23/13 We started growing up cholera overnights to run our biofilm degredation assay with our newly purified DspB and AmyA. Samples 1-24 were treated at T = 0 to test for enzymatic inhibition of biofilm growth. Samples 25-48 were treated at T = 48 to test for enzymatic degradation of the biofilm. Treatments were administered according to the following: Biofilm Degradation Overnights 1-3 Control 4-6 Urea 7-9 2.5 uL DspB 10-12 12.5 uL DspB 13-15 25 uL DspB 16-18 2.5 uL AmyA 19-21 12.5 uL AmyA 22-24 25 uL AmyA 25-27 Control 28-30 Urea 31-33 2.5 uL DspB 34-36 12.5 uL DspB 37-39 25 uL DspB 40-42 2.5 uL AmyA 43-45 12.5 uL AmyA 46-48 25 uL AmyA
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