Team:Paris Bettencourt/Notebook/TMAU

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Notebook

Trimethylaminuria

July

July 3rd

We made a PCR of our tmm gBlock using this protocol:

ReagentVolume
1X4X
5X Phusion HF Buffer20uL80uL
10mM dNTPs2uL8uL
oPB.010 (forward primer)1uL4uL
oPB.012 (reverse primer)1uL4uL
DNA1uL4uL
DMSO3uL12uL
Phusion polymerase1uL4uL
Nuclease Free Water71uL284uL

We used this thermocycle:

TemperatureTime
Start98°C30s
Cycle x3598°C10s
Cycle x3552°C30s
Cycle x3572°C30s
Finish72°C5min
Blind10°C-

After gel migration, we got this:

The PCR worked.

July 10th

1) Mini prep to extract plasmids (Qiagen)

I used this protocol. I made 2 tubes and got 163.2 and 242 ng/uL as concentrations.

2) Digestion of miniprep product with EcoR1 & Pst1

Tube 1 (163ng/uL)Tube 2 (242ng/uL)
pSB1C3 from miniprep22uL30uL
10X FD Buffer5uL5uL
PstI2.5uL2.5uL
EcoRI2.5uL2.5uL
NF Water18uL10uL

3) Gel migration

I had the digested pSB1C3 run on a gel using 0.5X TBE (2H-50V). We clearly saw a band at 2kb corresponding to pSB1C3. I cut the gel and stored it at 4°C.

July 11th

1) Gel purification of digested pSB1C3

I used this protocol

Result: I got concentrations of 59.6 and 55.1 ng/uL.


2) Ligation of digested tmm and digested pSB1C3

I used NEBiocalculator to calculate the mass of insert and vector I had to put (Insert length: 1488 bp, Vector length: 2037 bp, Vector DNA mass: 0,3 ug, Ratio insert:vector: 2:1). I need to put 450ng of insert and 300 ng of vector.

Volume
Insert (tmm)20uL of 10.1 ng/uL and 20uL of 13.7 ng/uL
Vector (pSB1C3)5uL of 59.6ng/uL
T4 Ligase3uL
T4 Ligase Buffer6uL
NF Water6uL

I incubated overnight at room temperature

July 12th

We transformed NEB and MG1655 with our ligation product according to this protocol

We finally had no colonies.



July 14th


July 15th

1) Gel extraction of pSB1C3 digested plasmid

I used this protocol. I finally got 2 tubes with 13.9 and 19.8ng/uL concentrations.


2) PCR purification

I purified the PCR product of July 14th using this protocol.


3) Ligation of tmm PCR product and pSB1C3

I used NEBiocalculator to to calculate the needed amount of insert and vector with the values:

  • Insert length: 1488 bp
  • Vector length: 2037 bp
  • Vector DNA mass: 50 ng
  • Ratio insert: I made 1 ligation of ratio 1:1, 2:1 and 3:1 (insert:vector).

I made the ligation with the 13.9 ng/uL concentrated insert tube. I used this protocol:

1:12:13:1
Digested tmm (insert)1.5uL3uL4uL
Digested pSB1C3 (vector)4uL4uL4uL
T4 Ligase1uL1uL1uL
T4 Ligase Buffer4uL4uL4uL
Nuclease Free water9.5uL8uL7uL

I incubated the tubes at 24°C for 30min.


4) Transformation into NEB and MG1655

I used the Heat Shock transformation protocol to transform NEB and MG1655 with each one of the ligation products with ratios 1:1, 2:1 and 3:1 (I got 6 plates).



July 16th

1) Results of the transformations

We got no colonies in MG1655, big and small colonies for 1:1 and 2:1 ratios and only big colonies with 3:1 ratios. I prepared 5 liquid cultures in LB+Chloramphenicol: 1 for each type of colony for 1:1 and 2:1 ratios, and 1 for big colonies for 3:1 ratio. I incubated them at 37°C.


2) PCR colony

To check if the colonies developped in my plates have tmm gene, I made a PCR with this protocol and I ran a gel. I got nothing on it. I will do miniprep and digestion on July 17th to identify tmm construct into pSB1C3.



July 17th

1) Miniprep using each liquid culture of NEB+tmm-pSB1C3

I used this protocol.

2) Digestion of the plasmids by EcoRI

Then I digested the miniprep product by EcoRI in order to get a linearised plasmid before running it on a gel.

ReagentVolume
Nuclease free water12uL
10X Fast Digest Buffer2uL
plasmid DNA5uL
EcoRI1uL
TOTAL20uL

I incubated the tubes at 37°C during 30min.


3) Gel

I ran on a gel each one of the tubes (small and big colonies from 1:1 and 2:1 ratios and big colony from 3:1 ratio). But the plasmids may have not been digested enough because I got many bands.



July 18th

I will try again to identify tmm gene in my transformed bacteria.

1) Digestion of plasmids with EcoRI and PstI.

This time I digested by EcoRI and PstI to be able to directly identify tmm gene on the gel. I nanodroped the miniprep product from 17th. We need 1ug of DNA for the digestion.

Concentration (ng/uL, given by Nanodrop)Needed volume (uL)
big 1:1 ratio colony316.43.5
small 2:1 ratio colony156.97
big 2:1 ratio colony207.65
big 3:1 ratio colony207.64

Digestion protocol:

Reagentbig 1:1 ratio colonysmall 2:1 ratio colonybig 2:1 ratio colonybig 3:1 ratio colony
Nuclease free water13.5uL10uL12uL13uL
10X Fast Digest Buffer2uL2uL2uL2uL
plasmid DNA3.5uL7uL5uL4uL
EcoRI0.5uL0.5uL0.5uL0.5uL
PstI0.5uL0.5uL0.5uL0.5uL
TOTAL20uL20uL20uL20uL

1)Gel

I ran all the samples on a gel but I did not get the bands I expected. We have to try a new cloning.



July 22nd

I had prepared liquid cultures to be able to do minipreps.

1) Miniprep of liquid cultures to get pSB1C3

I used this protocol. The Nanodrop gave me a concentration of 317ng/uL.


2) Digestion of pSB1C3 and tmm PCR product by EcoRI and PstI.

  • pSB1C3:
    ReagentVolume
    Nuclease free water25uL
    10X Fast Digest Buffer5uL
    plasmid DNA14uL
    EcoRI2.5uL
    PstI2.5uL
    Fast AP (phosphatase)1uL
    TOTAL40uL

  • tmm PCR product
    ReagentVolume
    Nuclease free water27uL
    10X Fast Digest Buffer5uL
    plasmid DNA13uL
    EcoRI2.5uL
    PstI2.5uL
    TOTAL50uL

3)Gel

For pSB1C3 we can see two bands corresponding to 900bp and 2000bp (backbone) fragments. I will be able to extract the 2000bp fragment. But I got nothing for tmm.


4)Gel extraction

I used this protocol.

July 29th

We need tmm PCR product to be able to do a new cloning.

1) PCR of tmm gBlock

ReagentVolume
1X
5X Phusion HF Buffer10uL
10mM dNTPs1uL
oPB.010 (forward primer)0.5uL
oPB.012 (reverse primer)0.5uL
DNA1uL
DMSO1.5uL
Phusion polymerase0.5uL
Nuclease Free Water35uL

We used this thermocycle:

TemperatureTime
Start98°C30s
Cycle x3598°C10s
Cycle x3556°C for tube 1, 51°C for tubes 2 and 330s
Cycle x3572°C1min
Finish72°C5min
Blind10°C-

2) Gel

No bands were visible, even for the ladder. I tried a new one and got a few tiny bands for tube 3, particularly at 1500bp. I tried a gel extraction using this protocol and got 4.7ng/uL (unusable). I tried otherwise:


3) PCR purification

I puridied tube 3 according to this protocol and got a concentration of 11ng/uL which was not enough to do a digestion.


4) New PCR

ReagentVolume
1X
5X Phusion HF Buffer10uL
10mM dNTPs1uL
oPB.010 (forward primer)0.5uL
oPB.012 (reverse primer)0.5uL
DNA1uL
DMSO1.5uL
Phusion polymerase0.5uL
Nuclease Free Water35uL

We used this thermocycle:

TemperatureTime
Start98°C30s
Cycle x3598°C10s
Cycle x3555°C30s
Cycle x3572°C1min
Finish72°C5min
Blind10°C-

3) PCR purification

I puridied tube 3 according to this protocol.

August

August 1st

1)Digestion of purified PCR product

ReagentVolume
Nuclease free water20uL
10X Fast Digest Buffer20uL
plasmid DNA7uL
EcoRI0.5uL
PstI0.5uL
TOTAL50uL

I incubated the digestion at 37°C during 1h.

2) Purification of digested tmm

I used this protocol and got a really low concentration, unusable for ligation.

3) New PCR

I prepared 4 tubes according to this protocol:

ReagentVolume
1X
5X Phusion HF Buffer10uL
10mM dNTPs1uL
oPB.010 (forward primer)0.5uL
oPB.012 (reverse primer)0.5uL
DNA1uL
DMSO1.5uL
Phusion polymerase0.5uL
Nuclease Free Water35uL

We used this thermocycle:

TemperatureTime
Start98°C30s
Cycle x3598°C10s
Cycle x3551°C30s
Cycle x3572°C1min
Finish72°C5min
Blind10°C-

4) Gel

Nothing was visible on the gel.


5) New PCR

ReagentVolume
1X
5X Phusion HF Buffer20uL
10mM dNTPs2uL
oPB.010 (forward primer)5uL
oPB.012 (reverse primer)5uL
DNA1uL
DMSO3uL
Phusion polymerase2uL
Nuclease Free Water53uL

We used this thermocycle:

TemperatureTime
Start98°C30s
Cycle x3598°C10s
Cycle x3550°C30s
Cycle x3572°C45s
Finish72°C5min
Blind10°C-

6) Gel

There was a diffused band next to 200bp. Maybe there is a problem of specificity of the primers. We will try a PCR with the very first protocol which worked.


August 5th

1) PCR of tmm gBlock

ReagentVolume
1X
5X Phusion HF Buffer20uL
10mM dNTPs2uL
oPB.010 (forward primer)1uL
oPB.012 (reverse primer)1uL
tmm DNA1uL
DMSO3uL
Phusion polymerase1uL
Nuclease Free Water71uL

We used this thermocycle:

TemperatureTime
Start98°C30s
Cycle x3598°C10s
Cycle x3552°C30s
Cycle x3572°C30s
Finish72°C5min
Blind10°C-

We still got nothing. We decided to design new primers.
August 6th

We think that that the reverse primer (oPB012) might have a strong secondary structure and that it may prevent the PCR to work as expected. We designed modified versions of oPB010 and oPB012 respectively called oPB056 and oPB057, shorter then the first ones. We removed the first restriction site at each end making sure the other enzyme would still have enough nucleotides to bind. We also shortened the bind part to have a melting temperature around 52°C.


oPB.010 TATAGAATTCGCGGCCGCTTCTAGAGCTGACAGCTAGCTCAGTCCTAG
-> oPB.056 CTTCTAGAGCTGACAGCTAGCTCAGTCC

oPB.012 TTAACTGCAGCGGCCGCTACTAGTATCAGTGGTGATGGTGATGATGGTTA
-> oPB.057 TACTAGTATCAGTGGTGATGGTGATGATG

Note:The digestion should now use XbaI and SpeI as EcorI and PstI were removed from the PCR product.

August 13th

We received our new primers (oPB.056 and oPB.057).


1) New PCR with different combinations or primers

We used this protocol:

ReagentTube 1Tube 2Tube 3Tube 4
5X Phusion HF Buffer20uL20uL20uL20uL
10mM dNTPs2uL2uL2uL2uL
tmm DNA10L10L10L10L
DMSO3uL3uL3uL3uL
Phusion polymerase2uL2uL2uL2uL
Nuclease Free Water53uL53uL53uL53uL
oPB.0105uL-5uL-
oPB.0125uL--5uL
oPB.056-5uL-5uL
oPB.057-5uL5uL-
TOTAL100uL100uL100uL100uL

We used this thermocycle:

TemperatureTime
Start98°C30s
Cycle x3598°C10s
Cycle x3552°C30s
Cycle x3572°C30s
Finish72°C5min
Blind10°C-
Date 2

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September

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October

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