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Team:OU Norman/Project/Notebook/Main
From 2014.igem.org
May-20-2014 Transformation of KIVD + PUC19
Purpose: Uptake PUC19 plasmid which contains KIVD with iGEM restriction sites into chemically competent E. coli cells. Then, the cells can be prepped for freezer stocks. Methods: Bacterial transformation Results: All plates had hundreds of white colonies
May-23-2014 DNA Extraction of mlsR + 4P #13
Purpose: Remove plasmid from E. coli cells Methods: Preformed DNA extraction via AquaPlasmid protocol. I amended the protocol to use as much cell mass that was obtainable from cultured plates. Results: 102.8 ng/µl of DNA extracted
May-23-2014 DNA Extraction of Promoters and mlsR + 6D
Purpose: Extract plasmids containing respectively promoters 20E and 20A, as well as mlsR + Terminator (6D) Methods: AquaPlasmid DNA Extraction. The amendments include 400 µl of AquaPlasmid, 250 µl of supernatant, 1250 µl of 70% isopropanol, and 0.5 mL of 70% ethanol Results: One successful extraction of 82.4 ng/µl was attained for mlsR + 6D.
May-27-2014 DNA Extraction of mlsR + 4P & 20A
Purpose: Extract mlsR + (Terminator) 4P. mlsR contains erythromycin resistance, whereas 4P is the terminator. Methods: AquaPlasmid DNA Extraction. I extended all times for things such as centrifuging, vortexing. Results: mlsR + 4P again gave me a successful result of 87.4 ng/µl. This leads me to believe that the problem with extracting the plasmids containing the promoter is in itself flawed.
May-28-2014 Plasmid Extraction
Purpose: Extract plasmids contain the respective parts; promoters, mlsR + terminator, TER + terminator, and KIVD. Methods: Using QIAprep Spin Miniprep Kit (250) following the provided instructions, the following plasmids were obtained. We discussed the PCR purification filters will also work for the plasmid extraction kit. Results: mlsR + terminator, as well as the promoters had a successful extraction range from 200-400 ng/µl. However, KIVD failed to produce adequate results.
May-29-2014 Digestion of Promoters
Purpose: It is necessary to screen for an insert in our promoter plasmid to ensure that the part we need is actually there through the means of visible confirmation Methods:
| Sample | Amount |
| Promoter | 500 ng |
| EcoR1 | 1 µl |
| Pst1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
Place in heat bath for 45 minutes at 42°C Immediately place on heat block for 15 minutes at 80°C to deactivate restriction enzymes Ready for gel electrophoresis
May-29-2014 Gel of Promoters
Purpose: Make 2% agarose gel in order to look at the bands, which is a reflection of the linearized base pair size Methods:
| Lane 1 | 1 KB Plus |
| Lane 2 | 20A #15 |
| Lane 3 | 20E #11 |
| Lane 4 | 20E #14 |
| Lane 5 | 23B #3 |
| Lane 6 | 1KB Plus |
Results: Even though promoters are not represented by any visible bands, they may still be there. Therefore, 3A assembly of promoters + Adh6 and KIVD + terminator are to be conducted
May-30-2014 Plasmid Extraction of KIVD
Purpose: The plasmid containing KIVD needs to be extracted so the KIVD gene can be excised and used for future ligations. Methods: QIAprep Spin Miniprep Kit (250) on KIVD #1 and #2 Results: KIVD #1 had a sufficient DNA extraction of 77.7 ng/µl, whereas the second KIVD sample was at 63 ng/µl. These results are sufficient, but more DNA would be ideal.
June-2-2014 Digestion of KIVD
Purpose: KIVD needs to be digested in preparation of a double ligation with a terminator and backbone. Methods:
| Sample | Amount |
| Promoter | 500 ng |
| EcoR1 | 1 µl |
| Pst1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
Place in heat bath for 45 minutes at 42°C Immediately place on heat block for 15 minutes at 80°C to deactivate restriction enzymes Ready for gel and 3A
June-2-2014 Digest of mlsR + terminator and Ter + terminator
Purpose: Digest out parts to be ligated together and placed into a different backbone to selectively choose transformant with new antibiotic resistance.
| Sample | Amount |
| mlsR + terminator | 500 ng |
| Xba1 | 1 µl |
| Pst1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
| Sample | Amount |
| Ter + terminator | 500 ng |
| EcoR1 | 1 µl |
| Spe1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
Also, digest Ter and mlsR to compare to the 3As
Place in heat bath at 42°C fir 45 minutes Place in heat block at 80°C for 15 minutes
June-2-2014 Gel of mlsR + terminator, Ter + terminator, and KIVD
Purpose: check for correct size of anticipated inserts Methods:
| Lane 1 | 1 KB Plus |
| Lane 2 | Ter + 4P #5 |
| Lane 3 | Ter + 6D #13 |
| Lane 4 | Ter #4 |
| Lane 5 | 1 KB Plus |
| Lane 6 | mlsR + 6D #9 |
| Lane 7 | mlsR #24 |
| Lane 8 | mlsR + 6D #12 |
| Lane 9 | mlsR #24 |
| Lane 10 | mlsR + 4P #13 |
| Lane 11 | mlsR #24 |
| Lane 12 | mlsR + 4P #16 |
| Lane 13 | 1 KB Plus |
| Lane 14 | KIVD #1 |
| Lane 15 | KIVD #2 |
| Lane 16 | 1 KB Plus |
Results: No inserts found for mlsR + terminator or KIVD
June-3-2014 Plasmid Extraction of KIVD, (Ter + 4P)
Purpose: Extract plasmids containing KIVD and a second plasmid extraction containing Ter + 4P Methods: QIAprep Spin Miniprep Kit (250) Results: Both plasmids were able to sufficiently extract 90 ng/ul of DNA
June-3-2014 Digest of KIVD and Ter + Terminator
Purpose: Excise parts of interest to be used to ligate with another part and plasmid Methods:
| Sample | Amount |
| KIVD | 500 ng |
| Xba1 | 1 µl |
| Pst1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
| Sample | Amount |
| mlsR + terminator | 500 ng |
| Xba1 | 1 µl |
| Pst1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
Place in heat bath at 42°C fir 45 minutes Place in heat block at 80°C for 15 minutes
Also digest Ter #4. The digest of Ter is to be compared to Ter + Terminator to verify correct insert size
June-3-2014 Gel of KIVD and Ter + Terminator
Purpose: Confirm insert size and presence via gel electrophoresis Methodology:
| Lane 1 | 1 KB Plus |
| Lane 2 | Ter + 4P #1 |
| Lane 3 | Ter #4 |
| Lane 4 | KIVD #1 |
| Lane 5 | 1 KB Plus |
Results
:June-3-2014 Digest of PUC19 samples
Purpose: Excise parts of interest to be used to ligate with another part and plasmid Methods:
| Sample | Amount |
| PUC19 | 500 ng |
| Xba1 | 1 µl |
| Pst1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
Place in heat bath at 42°C fir 45 minutes Place in heat block at 80°C for 15 minutes
June-17-2014 Anaerobic Media
Purpose: Clostridium acetobutylicum is a strict anaerobe. Therefore, the cells will only grow in the absence of oxygen or extremely low levels of oxygen. Rather than use a glove box, this method is an easy alternative because the serum bottles in which we use are plentiful and cheap, not to mention portable. Methodology: The procedure can be found the protocol section of the wiki. Results: Serum bottles that possess an anaerobic environment will be yellow, whereas oxygen exposure will turn the solution peach color. We tested a bottle and this is what we saw.
June-24-2014 DNA Digest and Gel
Purpose: Digest of Bktb Methodology:
| Sample | Amount |
| Bktb | 500 ng |
| EcoR1 | 1 µl |
| Pst1 | 1 µl |
| 10X Fast Digest Buffer | 5 µl |
| BSA | 1 µl |
| DI Water | Add to make 50 µl total reactions |
Results:The results indicated that the DNA was too large to be our gene of interest. Also, there was only one band, indicating that the procedure did not successfully produce what we desired. It could be the plasmid without our gene of interest.
August-5-2014 DNA Extraction of PAN1
Purpose: Clostridium acetobutylicum naturally degrades plasmids. However, if we methylate the plasmid we can avoid this error. This is where PAN1 which is a plasmid comes into play because it is responsible for methylating our plasmid before its insertion into C. acetobutylicum. Methods: QIAGEN miniprep kit Results: Sufficient number of plasmids was extracted. Next step is to co-transform the plasmid into E. coli cells.
September-10-2014 Gel: PAN1
Purpose: We transformed our shuttle vector into E. coli cells with PAN1, which is our methylating plasmid. We now are going to run a gel to see base-pair size differences and the number of bands. The methylates samples should have two bright bands (PAN1 & shuttle vector). Methodology: 1% Gel electrophoresis Results: Failed. Only one band appeared on each lane indicating the two plasmids were not co-transformed into E. coli cells.
September-12-2014 3A: KIVD, Ter, Bktb, Adh6
Purpose: All previous attempts with 3A's have failed. In the hopes that the previous 3A failures are because of technique I have meticulously prepared how we will go about this. Methodology: Procedure can be found in protocol section of the wiki. Results: Theoretical part combinations: -promoter + Adh6 in pSB1K3 -KIVD + terminator in pSB1A3 -promoter + Bktb in pSB1K3 -Ter + terminator in pSB1A3
September-17-2014 Colony PCR: 3A
Purpose: Transformants from previous 3A had the RFP excised. Now the question is whether our parts have been inserted. Colony PCR will amplify DNA, followed by a gel to confirm base pair size sum of two parts. Methodology: Procedure can be found in protocol section of the wiki. Results: Three of the 40 samples showed a positive result. By which I mean the bands were the appropriate size according to the expected base pair summation of the two parts.
September-19-2014 DNA Extraction: Promoter + Bktb
Purpose: Quantify the cultures of grown up cells that contained the positive results from the previous 3A colony. Methodology: QIAGEN Miniprep Kit Results: The quantifications were successful and sufficient to send off for sequencing.
