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7/1/13
We made new cholera overnights, seeded from the overnights done on 6/24/13. New overnights were placed in the 30°. We also ran PCR on our B. subtilis purified DNA, we will run our product on gel on 7/3.
We heard back from the France iGem team today, they sent out our DspB sample, it should be here in about a week.
We prepared another 96-well plate according to the tables below:
96-Well Plate Setup
| Treatment:
|
| Blank Control
| Biofilm
| Urea
| Biofilm w/ CV
| Biofilm w/ CV
| Urea w/ CV
| Urea w/ CV
|
| Column:
| H | G | F | E | D | C | B | A
|
Master Solutions
| Row
| Growth Media
| Amount of Media (mL)
| Amount of V. cholerae (uL)
| Percent V. cholerae
|
| 1 | SLB | 1.4 | 14 | 1
|
| 2 | SLB | 1.4 | 14 | 1
|
| 3 | SLB | 1.4 | 28 | 2
|
| 4 | SLB | 1.4 | 28 | 2
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| 5 | SLB | 1.4 | 56 | 4
|
| 6 | SLB | 1.4 | 56 | 4
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7/3/13
We ran our B. subtilis PCR product and the AmyA cloning product on gel to check them. The B. subtilis showed no PCR product. We will need to re-purify our dna template and try the PCR again. Whitney has results for AmyA.
We took a plate reading and got a fairly consistent reading, however we are still having trouble washing the biofilm without disturbing it and losing some in the wash. To correct this, we will try to do the washes and prep work in Eppendorf tubes, spinning them down between washes to allow easy and precise removal of liquid without removing the biofilm as well. The samples will then be transferred to the 96-well plate just for the plate reading. Another possibility is to find a way to centrifuge the entire 9-well plate in between each wash. We will check with Dr. Grose on these on 7/5 and decide on which method we will use.
From our plate reading today, the 0.03% CV stain worked well and gave us a good read. We will use this rather than the 0.3% CV solution cited in our original protocol. Also, the 1% and 4% biofilms showed significantly different readings, however, both groups were fairly consistent within the groups. We decided that we will just run the 4% biofilms from now on to have standardized samples for more consistent data.
7/5/13
Today we discussed the possible methods of preventing biofilm in our washes of the 96-well plate. We will use the plate centrifuge in Dr. Grose’s lab to pellet our biofilm in the plate between each wash. We will also run a few samples with Eppendorfs to see if either method is more consistent.
7/8/13
We reran the PCR cleanup kit on our B. subtilis strain. We also received the DspB part from the France iGem team so we set up phusion PCR for the DspB using the BI107(forward) and BI108(Reverse) primers.
We also prepared a new 96-well plate according to the tables below:
96-Well Plate Setup
| Treatment:
| Blank Control
| Biofilm
| Urea
| Biofilm w/ CV
| Biofilm w/ CV
| Urea w/ CV
| Urea w/ CV
|
|
| Column:
| H | G | F | E | D | C | B | A
|
Master Solutions
| Row
| Growth Media
| Amount of Media (mL)
| Amount of V. cholerae (uL)
| Percent V. cholerae
|
| 1-6 | SLB | 8.8 | 352 | 4
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7/10/13
We ran the DspB PCR product on gel to check for the correct product. Get results from Whitney.
We also ran the digestion enzymes for Savinase.
We set up AmyA for sequencing to check to make sure that the plasmid was taken up correctly. Whitney has the results.
We also ran our 96-plate we prepared on 7/8/13. For the washes, we briefly centrifuged the plate in between the washes to help prevent the removal of the biofilm through the washes. However, our data was still very inconsistent. In some cases we saw a higher biofilm reading in the untreated samples than those treated with urea; this is the exact opposite of the result that we should have seen. In the two Eppendorf samples that we prepared, we saw a significant reduction of biofilm in the treated sample. After discussing our results with Dr. Grose, we decided to use Eppendorfs for the biofilm growth and treatment before transferring to a 96-well plate to take the reading. We will no longer grow the biofilms and treat them in the 96-well plate. This will also allow us to start new growths every day we come into the lab, allowing us to gather data more quickly and consistently.
After further discussion with Dr. Grose, we also decided that our biofilm assay protocol is ready to use. We now need to finish purifying our finished enzyme products to be able to test them. We will now focus on getting our enzymes cloned into E. coli and purified for testing.
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