Team:TU Delft/17 August 2010 content

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(→Alkane degradation)
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==Alkane degradation==
==Alkane degradation==
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====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:
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:
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|‘E - pSB1C3 - P’
|‘E - pSB1C3 - P’
|}
|}
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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 [https://2010.igem.org/Team:TU_Delft#page=protocols/agarose_gel 1% agarose gel]:
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[[Image:TU_Delft_Digestion_16-08-10.png|400px|thumb|left|1% agarose gel run at 100V for 1 hour. Of all samples 10 µL was loaded with 2 µL loadingbuffer. 2 µL was loaded of marker]]
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Lane description
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"
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|'''#'''
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|'''Description'''
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|'''Expected size (bp)'''
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|'''OK?'''
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|-
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|1
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|Smartladder
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|Varies
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|Yes
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|-
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|2
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|011A cut
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|<b>1523</b>, 1199, 857
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|Yes
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|-
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|3
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|013K cut
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|1825, <b>1592</b>, 1342, 43
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|Yes
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|-
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|4
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|017A cut
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|<b>1410</b>, 1199, 857
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|Yes
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|-
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|5
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|018A cut
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|1185, 872, <b>793</b>, 43
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|Yes
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|-
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|6
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|402A cut
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|1199, 857, <b>543</b>
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|Yes
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|-
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|7
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|403A cut
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|1185, 872, <b>397</b>, 43
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|Yes
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|-
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|8
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|pCaiF in pANY cut
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|1277, 1143, <b>74</b>
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|Yes
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|-
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|9
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|B0032 in pSB1A2 cut
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|1185, 872, <b>35</b>
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|Yes
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|-
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|10
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|B0032 in pSB1A2 cut
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|1185, 872, <b>35</b>
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|Yes
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|-
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|11
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|pAlkS cut
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|1272, 1138, <b>106</b>
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|Yes
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|-
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|12
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|pSB1C3 cut
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|<b>2035</b>, 1081
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|Yes (However incomplete digestion, see 3116 bp)
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|-
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|15
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|BioRad EZ Load Marker
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|Varies
 +
|Yes
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|}
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 +
====Ligation====

Revision as of 11:17, 18 August 2010

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:

1% agarose gel run at 100V for 1 hour. 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 011A cut 1523, 1199, 857 Yes
3 013K cut 1825, 1592, 1342, 43 Yes
4 017A cut 1410, 1199, 857 Yes
5 018A cut 1185, 872, 793, 43 Yes
6 402A cut 1199, 857, 543 Yes
7 403A cut 1185, 872, 397, 43 Yes
8 pCaiF in pANY cut 1277, 1143, 74 Yes
9 B0032 in pSB1A2 cut 1185, 872, 35 Yes
10 B0032 in pSB1A2 cut 1185, 872, 35 Yes
11 pAlkS cut 1272, 1138, 106 Yes
12 pSB1C3 cut 2035, 1081 Yes (However incomplete digestion, see 3116 bp)
15 BioRad EZ Load Marker Varies Yes

Ligation

Digestion

# Sample Enzyme 1 Enzyme 2 Enzyme 3 Buffer BSA Needed fragment
1 011A EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - J23100-J61100-alkB2-J61100-rubA3 - S’
2 013K XbaI PstI HindIII NEBuffer 2 ✓ ‘X - J61100-rubA4-J61100-rubR-B0015 - P’
3 017A EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - J23100-J61100-ladA - S’
4 018A XbaI PstI AseI NEBuffer 2 ✓ ‘X - J61101-ADH - P’
5 402A EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - J23100-J61101-PhPFDα - S’
6 403A XbaI PstI AseI NEBuffer 2 ✓ ‘X - J61101-PhPFDβ - P’
7 pCaiF EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - pCaiF - S’
8 B0032 XbaI PstI AseI NEBuffer 2 ✓ ‘X - B0032 - P’
9 B0032 XbaI PstI AseI NEBuffer 2 ✓ ‘X - B0032 - P’
10 pAlkS EcoRI SpeI AseI NEBuffer 2 ✓ ‘E - pAlkS - S’
11 pSB1C3 EcoRI PstI None NEBuffer 2 ✓ ‘E - pSB1C3 - P’

The digestions were executed for 15 minutes at 37 degrees, and checked on 1% agarose gel:

1% agarose gel run at 100V for 1 hour. 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 011A cut 1523, 1199, 857 Yes
3 013K cut 1825, 1592, 1342, 43 Yes
4 017A cut 1410, 1199, 857 Yes
5 018A cut 1185, 872, 793, 43 Yes
6 402A cut 1199, 857, 543 Yes
7 403A cut 1185, 872, 397, 43 Yes
8 pCaiF in pANY cut 1277, 1143, 74 Yes
9 B0032 in pSB1A2 cut 1185, 872, 35 Yes
10 B0032 in pSB1A2 cut 1185, 872, 35 Yes
11 pAlkS cut 1272, 1138, 106 Yes
12 pSB1C3 cut 2035, 1081 Yes (However incomplete digestion, see 3116 bp)
15 BioRad EZ Load Marker Varies Yes

Ligation

Following the digestion the products were ligated for 15 minutes at RT:

# 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’
7 014C ‘E - J23100-X-J61100-alkB2-J61100-rubA3 - S’ ‘X - J61100-rubA4-J61100-rubR-B0015 - P’ ‘E - pSB1C3 - P’ (Pre-shipped linearized plasmid)

The ligations were performed in parallel: one batch using the NEB T4 DNA ligase and buffer, the other batch the Fermentas T4 DNA ligase and buffer. We hope to be able to discern the differences in efficiency between the two (if there are any). To test whether the ligations were succesful, i uL of ligation mix was used as template for a PCR amplification. The results will be known by tomorrow.

Transformation

The ligation products (19 uL) were used to transform 25 uL of commercially competent TOP10 cells (OneShot from Invitrogen) in accordance with the OneShot protocol. In order to test the background the digestion product of pSB1C3 was transformed (digestion control) as well as the ligation controls.

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