Team:TU Delft/17 August 2010 content


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Alkane degradation


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:

1% agarose gel run at 90V for 50 min. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker

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.


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.