Team:RHIT/Notebook/Journal

Inoculated a colony of E. coli that expressed the alpha mating factor (JW2004-1) in LB broth incubated in shaker at 37°C for 24 hours.

Plated the Blue construct and Red constructs (pRS413A and pRS416A) on LB + Amp and incubated at 37°C for 24 hours.

Inoculated 3 Falcon tubes with 3 ml of LB broth and isolated colonies from the following plates from 6/13: Blue construct, pRS413A, and pRS416A.

Miniprep four overnight cultures of the following for plasmid extraction: E. coli expressing alpha mating factor, the blue construct, and the red constructs pRS413A and pRS416A.

Streak untransformed JW2004-1 E. coli (on M9 minimal media, M9 minimal media +his M9 minimal media +lac, M9 minimal media +his +lac). Observe the growth 24 hours after plating to determine whether or not strain is in fact -his.

Observation: It was expected to see growth on the +his and +his +lac plates, with no growth on the +lac and minimal media plates because of the deletion of the his gene. Strains appear to be growing the same on all medias; however a plating error occurred and LB broth from the inoculated culture of JW2004-1 was streaked on the plates along with the cells, which may create a "false positive" by giving the cells access to histidine. Colonies were picked from the plated JW2004-1 and resuspended in a 5X M9 salt solution prior to plating to wash away LB broth. The resuspensions were plated again on the M9 medias mentioned before plus kanamycin media (to ensure that the strain is Kan resistant as it should be), and the results will be observed tomorrow, 6/26. NEB5α subcloning efficiency cells were also plated with the three colonies.

Finished preparing the SOC media. Two separate 20 mL aliquots were made. 0.025 g of lactate in one of the aliquots to make SOC+lac.

Prepare competent cells for transformation. Produced thirteen 1.0 mL aliquots of competent cells.

Hypothesis: Lactate is brought into the cells and stored because lactate is potential fuel source for JW2004.

Need to determine if alpha mating factor is on the surface of the cells. We need to work on the Blue construct and E. coli construct at the same time.

We made selection media for the selection of alpha construct transformants.

The new selection scheme will employ three types of media and a positive and negative control.

Groups: (-) control- trans w/ water; trial- trans w/ alpha plasmid; (+) control- trans w/ pRS413

4 of the 8 groups were outgrown with lactate in the SOC.

Each of the 8 groups of competent cells will be spread plated onto the selectio media. Wash cells with M9 salts solution before spread plating onto selective media.

Media: M9 Minimal; M9 Minimal + Lactate; M9 Minimal + Histidine

Expected outcome: We expect the negative control to grow on M9 Minimal + Histidine. The negative control should not grow M9 Minimal and M9 Minimal + Lactate. The trial group will grow on M9 Minimal + Lactate and M9 Minimal + Histidine. The trial group will not grow on M9 Minimal. The positive control will grow on all media.

Performing a digest on the Blue construct and pRS416. We are doing a double digest with Xba and Spe-HF.

Run digest products on gel to confirm success of digest.

Gel: 9% agarose poured at 8:09 a.m.

Samples: 10 µL digested DNA, 8 µL water, 1.1 µL 10X loading dye for all. Undigested DNA for control.

We performed the transformant selection protocol. The media was incorrect. Sucrose was used as the carbon source.

The SOC+lac used in the outgrow stage may have been contaminated. We will not use the results.

Setup digest to cut blue construct and pRS416:

*Used the following combinations of enzymes:
Xba1 and Spe1 for pBlue and pRS416
EcoR1 and Spe1 for pBlue and pRS416

• 7 µL water
• 1 µL 10X Cutsmart Buffer
• 1 µL DNA
• 1 µL Enzyme

Incubate for 1-2 hours at 37°C.

Heat inactive Xba1 at 65°C for 20 minutes.

Double reaction size and add Spe-1HF. Incubate for 1-2 hours at 37°C.

Freeze digestion products

Run digestion products on gel to assess digest and remove digest DNA for ligation

Samples: 10.0 µL digestion product, 8.0 µL water, 1.1 µL loading dye

Start: 8:42 a.m. Stop: 9:50 a.m.

Extract fragments using Qiagen Quick Gel Extraction Kit to extract Blue construct and pRS416 with EcoR1 and Spe1.

Run gel to determine success of extraction and ratio for ligation.

20 mL 0.9% agarose gel (0.18 g agarose, 20 mL TBE)

Gel was inconclusive.

Repeat digest with more plasmid DNA.

Two additional DNA digests were done. One done with a higher concentration of DNA to achieve enough DNA for gel extraction and ligation

Digest 1: 7 µL water, 1.0 µL 10X buffer, 1 µL DNA, 1 µL enzyme; heat inactivate enzyme, add 8 µL water and 1 µL buffer, add 1 µL second enzyme and incubate again.

Digest 2: 1 µL 10X buffer, 8 µL DNA, 1 µL enzyme; heat inactivate enzyme, add 8 µL water, 1 µL buffer, and 1 µL second enzyme and incubate again.

Run gel to get bands for extraction.

Extract DNA from gel.

Remake M9 selection media for selection of alpha transformants.

Prepare chemically competent cells with JW2004. Use TSS Buffer to resuspend cells.

Transform JW2004 competent cells with E. coli construct.

Outgrow in SOC and SOC+lactate.

Spread plate onto M9 selection medias.

Diagnostic test on pRS416 with EcoR1, Xba1, Pst1, and EcoR1/Pst1 to determine if pRS416.

Run on gel to visualize results.

Transform JW2004 competent cells with E. coli construct.

Outgrow in SOC.

Plate onto antibiotic selection media.

Determine correct concentration of lactate to make M9+lactate selection media.

Digest blue construct with Spe-HF.

Run linearized digestion products on gel.

Transform JW2004 with E. coli construct.

Concentration of antibiotic in selection media:

• Amp-50 microgram/mL
• ChL-170 microgram/mL
• Kan-50 microgram/mL

Add antibiotic after cooling sterile media. Antibiotic must be sterile.

Plate ligation products.

Run gel to assess efficiency for diagnostic digest.

Boil prep ligation transformants for diagnostic digest.

Run boil lysis products on gel for visual confirmation of ligation.

Results were inconclusive.

Transform JW2004 with E. coli construct.

Transformation was inconclusive.

Transform JW2004 with E. coli construct.

Transformation was inconclusive.

Make fresh M9+lactate selection media.

Prepare fresh chemically competent cells.

Run ligation products on gel for analysis.