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Team:TU Delft/17 August 2010 content
From 2010.igem.org
Contents |
Alkane degradation
Digestion
Yesterday's transformed competent cells of the 014C, 020C, 405C, 327C and 304C ligation mixes (using the BioBrick assembly method) yielded only red colonies, indicating the lack of positive colonies. It is interesting to note the presence of numerous red colonies on the digest control plate, whereas the ligation control plate (Digested pSB1C3 + ligase buffer + ligase) yielded but 1 red colony (at similar trasnsformation mix concentrations). Based on the information gathered thus far a few hypotheses were made:
Hypothesis I: The ligase buffer inhibits transformation efficiency in some way.
Hypothesis II: The ligation time of 20 mins was too short.
In order to test hypothesis I, the following mixes will be transformed into commercial competent cells:
| # | Fragment 1 | Ligase buffer? | Total volume (uL) |
| 1 | E-pSB1C3-P (2 uL) | No | 20 |
| 2 | E-pSB1C3-P (2 uL) | Yes (2 uL) | 20 |
In order to test hypothesis II, a ligation kinetics assay was performed using E and P digested pSB1C3 as vector and J04450 as insert. The ligations were run for 30, 60, 120 and 240 minutes.
Furthermore, a second attempt was made at creating BioBricks 014C, 020C, 405C, 327C and 304C. pSB1C3 was digested additionaly using HindIII to cut the J04450 insert as follows:
| # | Sample | Enzyme 1 | Enzyme 2 | Enzyme 3 | Buffer | BSA | Needed fragment |
| 1 | pSB1C3 | EcoRI | PstI | HindIII | NEBuffer 3 | ✓ | ‘E - pSB1C3 - P’ |
The digestion mix was incubated at 37 degrees for 1 hour and at 80 degrees for 20 minutes to inactivate the restriction enzymes. The mixes were checked on 1% agarose gel:
Lane description
| # | Description | Expected size (bp) | OK? |
| 1 | Smartladder | Varies | Yes |
| 2 | pSB1C3 E+P digest | 2035, 1081 | Yes |
| 3 | pSB1C3 E+P+HindIII digest | 2035, 456, 403, 222 | Yes |
From the gel it could be concluded that the digestions were succesful.
Ligation
Following the digestion the products were ligated for 4 hours at 20 degrees and the ligase inactivated at 80 degrees for 20 min.:
| # | BioBrick | Fragment 1 | Fragment 2 | Destination Vector |
| 1 | 014C | ‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ | ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ | ‘E - pSB1C3 - P’ |
| 2 | 020C | ‘E - J23100-X-J61100-ladA - S’ | ‘X - J61101-ADH - P’ | ‘E - pSB1C3 - P’ |
| 3 | 405C | ‘E - J23100-X-J61101-PhPFDα - S’ | ‘X - J61101-PhPFDβ - P’ | ‘E - pSB1C3 - P’ |
| 4 | 327C | ‘E - pCaiF - S’ | ‘X - B0032 - P’ | ‘E - pSB1C3 - P’ |
| 5 | 304C | ‘E - pAlkS - S’ | ‘X - B0032 - P’ | ‘E - pSB1C3 - P’ |
| 6 | Ligation control | None | None | ‘E - pSB1C3 - P’ |
To test whether the ligations were succesful, 1 uL of ligation mix was used as template for a PCR amplification.
Alkane Degradation parallel attempt
Transformation
The ligation products (10 ul) from yesterday were used to transform 30 uL of TOP10 cells.
