Glasgow/Wetlab/Week10
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Week 10
Monday 3rd September 2007
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Christine's plates from 31/08/07 all grew except for 1B. Overnights were set up from 7 colonies on each of the plates on Sunday, labelled 1A1→1H7, 49 in total as there were none from plate 1B. Christine, Maia and Maija miniprepped them and digested them as follows:
This table is incorrect, see 04/09/07.PCR tube Colony DNA is taken from Insert Enzyme Expected sizes Result 1A D phzA→phzD EcoRI 3455bp, 18bp, 3654bp Negative 1B D phzA→phzD PstI 274bp, 6553bp Negative 1C S phzD→phzG EcoRI 7046bp, 18bp, 284bp Negative 1D S phzD→phzG PstI 1155bp, 2511bp, 3682bp Negative 1E S phzD→phzG PstI 1962bp, 1704bp, 3682bp Negative 1F Q phzD→phzG EcoRI 7046bp, 18bp, 284bp Negative 1G Q phzD→phzG PstI 1155bp, 2511bp, 3682bp Negative 1H Q phzD→phzG PstI 1962bp, 1704bp, 3682bp Negative
Tuesday 4th September 2007
- Christine realised that the site directed mutagenesis she had attempted on Thursday 30/08/07 was mistaken. S and Q were meant to be tested for phzA→phzD, and D was meant to be tested for phzD→phzG but S and Q were tested for phzD→phzG, and D was tested for phzA→phzD. This means the digests from 03/09/07 were also incorrect. However, all is not lost because the sequencing results for S and D came back today and they show that both S and D contain phzD→phzG. This means that, theoretically, digests of mutations 1C, 1D and 1E from 03/09/07 should be positive, but this is not the case.
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Before the sequencing results for S and D arrived, Christine set up site directed mutagenesis to correct that which she did 30/08/07. Used KOD and KOD_SDM programme as 30/08/07.
PCR tube Colony DNA is taken from Insert Primers used 1A D phzE→phzG EcoRI *E for + EcoRI *E rev 1B D phzE→phzG PstI *E for + PstI *E rev 1C D phzE→phzG PstI *F for + PstI *F rev 1D S phzA→phzD EcoRI *B for + EcoRI *B rev 1E S phzA→phzD PstI *C for + PstI *C rev 1F Q phzA→phzD EcoRI *B for + EcoRI *B rev 1G Q phzA→phzD PstI *C for + PstI *C rev After the PCR raction, these were each digested with DpnI for 1 hour at 37°C and PCR purified according to QIAGEN manual. After this they were transformed (15ul DNA into half a tube of TOP10 cells, heat shock of 30 seconds at 42°C, plated on carb) and labelled alphabetically according to PCR tubes above.
- Maija set up overnights in LB carb of colonies thought to contain phzA→phzD which were plated 23/08/07, labelled 1→10.
Wednesday 5th September 2007
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Maia, Maija and Christine miniprepped Maija's overnights of colonies thought to contain phzA→phzD. They were then digested with EcoRI and PstI.
Insert Enzyme Expected Sizes(5'→3') Expected Sizes (3'→5') Result phzA→phzD EcoRI 928bp, 2227bp, 18bp, 3654bp 1796bp, 1349bp, 3682bp No bands of interest phzA→phzD PstI 4757bp, 274bp, 1796bp 1796bp, 1349bp, 3682bp No bands of interest - Christine set up overnights from yesterday's site directed mutagenesis transformations, 7 colonies per plate were seeded, labelled A, B and C according to PCR tube (D, E, F, and G did not contain what they were originally thought to, see 04/09/07), and each colony labelled 1→7.
Thursday 6th September 2007
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Lynsey, Maia and Maija miniprepped Christine's overnights of colonies containing phzE→phzG which had undergone one round of site directed mutagenesis. They were the digested as follows:
Label Original Colony Insert Site Mutated Enzyme Expected Bands Result 1A D phzE→phzG EcoRI (679) EcoRI 7046bp, 18bp, 284bp No bands of interest 1B D phzE→phzG PstI (1688) PstI 2511bp, 3682bp, 1155bp No bands of interest 1C D phzE→phzG PstI (2237) PstI 1704bp, 1962bp, 3682bp No bands of interest
Friday 7th September 2007
Further work
Before proceeding with the XylR+Pr system we needed to confirm their presence in in TOPO vector. Restriction analysis shoved the wrong band sizes expected and thus proved that earlier attempts to clone this fragment had failed. For another attempt we returned to the MT2 template DNA which was used to isolate the XylR genes originally. We used the minipreps from MT2 made on the 27th of july and used the KOD PCR protocol (see Protocol 9) to amplify up the XylR+Pr fragment using the primers Pr_prefix and XylR_suffix. Two MT2 miniprep DNA sources were used and both done in triplicate. The PCR products (1a, b, c and 2a, b, c) proved to be the right size (2131 bp), so we proceeded to clone these solutions into TOPO 4.0 starting by purifying the PCR product (see Protocol 14) and this solution was then added to the TOPO cloning reaction (see Protocol 12) and transformed (see Protocol 13) into TOP 10 cells. Colonies were observed on nearly all plates and so overnights in liquid media containing the appropriate antibiotics were set up using 3 colonies from each plate where possible.
| Plate | colony names |
| 1c (150μl) | A B C |
| 1c (100μl) | D E F |
| 1b (150μl) | G H I |
| 2b (150μl) | J K L |
| 2c (100μl) | M N O |
| 2a (100μl) | P Q R |
| 2a (150μl) | S T U |
| 2b (100μl) | V W |
The following day these cultures were all miniprepped according to the alkaline lysis miniprep protocol ((see Protocol 18) and run on a 1% agarose gel at 50 V to determine which of the colonies have the desired insert Colonies with the desired insert: A C F I J K M N O P Q R S T U V
All of the positive colonies were then further examined using restriction digest analysis (see Protocol 7) The correct banding pattern was observed in virtually all tested colonies, but A and C were used further as these showed stronger banding and thus contained more DNA.
To enable the assembly of the XylR system a digest (see Protocol 7) using EcoRI and SpeI was set up to put XylR+Pr into construction vectors (4/6B and 3/20G) NB: at this stage XylR still contains a PstI site. At the same time the already assembled XylR responsive promoter Pu+RBS (3/20G) was digested (see Protocol 7) with SpeI and PstI to allow insertion of reporter genes found in the repository. LacZ (1/14K) and Renilla reniformis luciferase (3/12E) which were both cut using XbaI and PstI. Following the water bath step the samples were run out on a 1% agarose gel at 50 V and band sizes analysed (only LacZ did not digest properly) and subsequently cut from the gel and the DNA purified using the Qiagen gel extraction kit (see Protocol 11). Ligation (see Protocol 16) reactions were then set up:
- XylR+Pr A into 4/6B
- XylR+Pr A into 3/20G
- XylR+Pr C into 4/6B
- XylR+Pr C into 3/20G
- Luciferase(3/12E) into Pu+RBS (3/20G)
The ligations were left out on the bench overnight and transformed (see Protocol 13) into TOP 10 cells and left in incubator overnight.
Growth was observed on all plates and colony PCR was performed on 1 colony from each plate but with 2 different primer pair combinations (exept for Pu+RBS +3/12E only VF2 and VR) using an adjustment of the TOUCH 2 programme (see Protocol 9) with slightly higher initial melting temperatures to accommodate the relatively high Tm of the first primer pair:
- Pr_prefix and XylR_suffix
- VF2 and VR
The samples were then run on 1% agarose gels and all showed amplifications of the right size (XylR+Pr A and C ~2131 bp and Pu+RBS+ 3/12E ~1500 bp), with the amplifications being slightly larger in the lanes where the VF2 and VR primer combination was used.
Overnights in liquid media were then set up using 2 colonies from each plate (2 plates for each ligation).
To further confirm the results of the ligations truly were positive the cultures were miniprepped according to Qiagan miniprepkit protocol (see Protocol 5) and restriction digest (see Protocol 7) analysis was performed:
- Cultures containing XylR+Pr in both construction vectors: PstI and PvuI enzymes used
- Cultures containing Pu+RBS +3/12E in3/20G: EcoRV and PvuI enzymes used
Digests were run on 1% agarose gels at 50V, and correct band sizes were observed.
We then went on to attempt putting the XylR+Pr system in adjacent to the Pu+RBS+3/12E in 3/20G. WylR+pr in the 4/6B construction vector was used as the size of the 3/20G vector (2079 bp) is too close to the size of XylR+Pr (2131 bp) to allow for efficient cutting of the bands from the gel.
- Insert (XylR+Pr A and C) was cut with EcoRI and SpeI
- Destination (Pu+RBS +3/12E) was cut with EcoRI and XbaI
After confirmation of band sizes, the correct bands were cut from the gel and the DNA extracted according to the Qiagen gel extraction kit protocol (see Protocol 11). T4 ligation reaction mixtures (see Protocol 16) were set up
- XylR+Pr A into Pu+RBS+3/12E
- XylR+Pr C into Pu+RBS+3/12E
Ligations were left on the bench for 30 mins at room temperature, then transformed into TOP 10 cells (see Protocol 13 and placed in the incubator overnight. Growth was seen on all plates, and colony PCR with 2 different primer pairs was done on 2 colonies from each plate to confirm insert size using an adjustment of the TOUCH 2 programme (see Protocol 9) with slightly higher initial melting temperatures to accommodate the relatively high Tm of the first primer pair:
- Pr prefix and VR
- VF2 and VR
Colony PCR showed the correct sized bands (~3600 bp) so over nights in liquid media were set up using 2 colonies from each plate. The next day the cultures were miniprepped using the Qiagen miniprep kit (see Protocol 5). As the XylR+Pr still contains a PstI site allowing for selection of plasmids containing the XylR+Pr gene, a single digest using the PstI enzyme. Band sizes were correct and the construct was sent off for sequencing to prepare for potential testing of the system using a Renilla reniformis luciferase assay kit.
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