Team:UQ-Australia/Notebook

Bioaccumulation (Mercury) Project

02/10/09: Nanodrop Mer-plasmid miniprep and prepare primers for MerT and MerP

Nanodrop results:

Sample name Concentration (ng/uL) 260/280 M3 JM109 pSUTP. pG.pmt 11.58 1.62 M4 JM109 pG.pmt 51.10 1.78 M18 JM109 pSUTP 24.32 2.32

Primer preparation:

Prepare the stocks of primers with concentration of 9.6 pmol/uL. Eight different primers (MerT seq1, MerT seq2, MerP seq1, MerP seq2, MT seq1, MT seq2) were diluted, following the protocol:

Centrifuge samples at high speed for 30seconds. Add nuclease free water to each sample as recorded on the label of the primer tube received from Sigma-Aldrich to obtain a 100uM solution. Primer Water volume (uL) Molecular Weigh MerT seq1 160 5781 MerT seq2 149 5910 MerP seq1 154 6100 MerP seq2 164 5869 MT seq1 155 6250 MT seq2 159 6148 Prepare 20uL of 10uM solution for each primer from their 100uM stocks. Prepare 20uL of 9.6uM solution for each primers by add 1.92uL of their 100uM stock to 18.08uL water à Used for DNA sequencing. Preprare solutions for DNA sequencing:

3 samples with the listed component was prepared for sequencing.

iGEM1: 10uL pG.pmt , 1uL MT seq1 (9.6uM) and 1uL MT seq2 (9.6uM) iGEM2: 10uL pSUTp, 1uL MerT seq1 (9.6uM) and 1uL MerT seq2 (9.6uM) iGEM3: 10uL pSUTp, 1uL MerT seq1 (9.6uM) and 1uL MerT seq2 (9.6uM). We do not have the sequencing result , hence, pSUTp was chosen to continue the project.

02/10/09: Nanodrop Mer-plasmid miniprep and preparing MerT and MerP primers

Nanodrop results:

Sample name -- Concentration (ng/uL) -- 260/280

• M3 JM109 pSUTP. pG.pmt -- 11.58 -- 1.62
• M4 JM109 pG.pmt -- 51.10 -- 1.78
• M18 JM109 pSUTP -- 24.32 -- 2.32

Primer preparation:

Stocks of primers with concentration of 9.6 pmol/uL were prepared. Eight different primers (MerT seq1, MerT seq2, MerP seq1, MerP seq2, MT seq1, MT seq2) were diluted, with the following protocol:

Centrifuge samples at high speed for 30seconds. Add nuclease free water to each sample as recorded on the label of the primer tube received from Sigma-Aldrich to obtain a 100uM solution. Primer - Water volume (uL) - Molecular Weight

• MerT seq1 - 160 - 5781
• MerT seq2 - 149 - 5910
• MerP seq1 - 154 - 6100
• MerP seq2 - 164 - 5869
• MT seq1 - 155 - 6250
• MT seq2 - 159 - 6148
• Prepare 20uL of 10uM solution for each primer from their 100uM stocks.
• Prepare 20uL of 9.6uM solution for each primer by adding 1.92uL of the 100uM stock to 18.08uL water.
• Preprare solutions for DNA sequencing:

3 samples with the listed components were prepared for sequencing.

• iGEM1: 10uL pG.pmt , 1uL MT seq1 (9.6uM) and 1uL MT seq2 (9.6uM)
• iGEM2: 10uL pSUTp, 1uL MerT seq1 (9.6uM) and 1uL MerT seq2 (9.6uM)
• iGEM3: 10uL pSUTp, 1uL MerT seq1 (9.6uM) and 1uL MerT seq2 (9.6uM).

We do not have the sequencing result , hence, pSUTp was chosen to continue the project.

24/09/09: Nanodrop and running Agarose gel for mini prep products from 23/09/09

A 1% agarose gel was set up with 1g agarose and 100ml TE buffer. The gel was run at 70V for 1 hour and post stained with ethidium bromide before visualisation.

Lane 1: dying load (mixture of 5uL 1kb ladder + 1uL 6xLoading dye)

Lane 2: pSUTP. pG. pmt (mixture of 10uL M3 JM109 pSUTP. pG.pmt + 2uL 6x loading dye).

Lane 3: pG.pmt (mixture of 10uL M4 JM109 pG.pmt + 2uL 6x loading dye).

Lane 4: pSUTP (mixture of 10uL M18 JM109 pSUTP + 2uL 6x loading dye).

CLICK HERE for the picture of the gel (File 2).

23/09/09: Mini prep Mer-gene containing bacteria

A mini prep was done to collect the plasmids that contain MerT and Mer P genes from the bacteria being grown on 24/09/09.

Products were named corresponding to the plasmids expected , including M3 JM109 pSUTP. pG.pmt , M4 JM109 pG.pmt and M18 JM109 pSUTP.

CLICK HERE for mini prep protocol.

CLICK HERE for the result (Can u please put the link to 24/09/09 here?)

22/09/09: Growing Mer-containing Bacteria

Three strains of bacteria containing MerT and MerP genes, including M3 JM109 pSUTP. pG.pmt (AmpR + KanR), M4 JM109 pG.pmt (AmpR) and M18 JM109 pSUT (AmpR) , were culture overnight at 370C on shaker.

M18 JM109 pSUT : bacteria contains MerT and MerP gene without introns.

M4 JM109 pG.pmt : bacteria with plasmids containg both MerT and MerP with introns in the sequence.

M3 JM109 pSUTP. pG.pmt : bacteria containg both pSUTP and pG. pmt plasmid.

CLICK HERE for details of medium preparation (File 1).

Result: There was growth of bacteria in all 3 tubes. However, the culture in tube 3 was less cloudy than the other.

15/09/09 - Repeat Transformation of BBaI716101
The overnight transformation from 14/09/09 was unsuccessful as no growth was observed on plates. It is probable that the cell death plasmid component (Part BBa_P1010) is functioning normally as it was believed that this component was 'faulty'. The same protocol as on 14/09/09 was used for the transformation with an extra positive control for comparison. A well characterised plasmid PUC18 (ampicillin resistance) was used as the positive control.

14/09/09 - Transformation of Plate 1, Well C
PLasmid BBa_I716101 with Part BBa_P1010 was transformed into XL-10 Gold Ultracompetent cells via heat shock and left in ampicillin plates at 37C overnight. DNA from plate 1, well C was stored in the Green iGEM box ('Fiona').

02/09/09 - Running Gel for Standard 23
2 gels were set up: - A--->1%, 140mL gel.Undigested and digested controls. Undigested/digested plasmid BBa_J63010 (Sample A, B and C) - B--->1%, 80mL gel. Undigested and Digested BBa_J63010 (Sample D,E)

01/09/09 - Agarose Gel and Nanodrop

• Miniprepped BBa_J63010 plasmids ran on 1% Agarose gel (TAE buffer, pH8.5) at 200V, 70mA for 1hour. Post-stain: Ethidium Bromide. Click HERE for a picture of the gel. Lanes1,8 - empty, Lane2 - ladder, Lanes3-7 - Miniprepped DNA (colonies A-E)
  
• Miniprepped BBa_J63010 plasmids (tubes A to E) analysed on nanodrop. (Data printout in IGEM_Mercury notebook)
  
• New space created for BBa_J63010 tubes A to E: in "Fiona" freezer, UQ-Australia IGEM Team's Green box.
  
• In the afternoon, digests of samples BBa_J63010 A-E (Standard 23) were run with EcoRI, AgeI, SpeI and PstI enzymes. Unfortunately NgoMIV enzymes were not in stock in the freezer so this digest could not be performed.
• The Standard 21 plasmid BBa_I716101 was again digested with PstI. All samples were left in the 37 degrees water bath overnight.
  

28/08/09 - DNA miniprep
Mini prep DNA extractions performed for BBa_J63010 plasmid selected yesterday and incubated overnight.
Tubes stored in "Fiona" freezer, UQ-Australia IGEM Team's orange box.

EDIT (01/09/09): these tubes now in IGEM
Green Box ("Fiona" freezer, bottom shelf).

27/08/09 - Colony Picking
5 colonies were picked and grown in ?mL of LB Broth and were left in the 37C incubator overnight. Samples were labelled 'BBa_J63010 A,B,C,D,E'

26/08/09 - Transformation of Plasmid BBa_J63010 with part BBa_J04450
BBa_J04450 was found in plate 1, well C. Colonies were grown overnight and will be picked and grown tomorrow.

--->Digest Gel of BBa_I716101 with pGfa2cLac2 control
A 1% agarose gel was run with 1kb ladder and samples with EcoRI, XhoI, BglII and BamHI restriction enzymes. The gel visualisation (image here) indicates that only EcoRI was able to slice the plasmid; it is possible that the other restriction sites are not on the plasmid.

25/08/09 - In-gel Extraction and Overnight Digests
20 chloroamphenicol plates were prepared with LB agar. BBa_I716101 and control plasmid pGfa2cLac2 were each digested with EcoRI, XhoI, BglII and BamHI enzymes (total of 8 samples). Digests have been left to run overnight in the 37C waterbath.

24/07/09 - DNA extraction and electrophoresis
Mini prep DNA extractions performed for MS427-pBAD, MS427-pKKJ143 cultures incubated overnight.
--> run on ethidium bromide gel to confirm DNA production.
Click HERE for a picture of the gel.

23/07/09 - Transformations with AG43 plasmids
E. coli incubated overnight in duplicate under the following conditions:

Strain: MS427 

Plasmid: pBAD (empty plasmid -AG43) 

- 2mL LB broth 

- 2µL ampicillin 

Strain: MS427 

Plasmid: pKKJ143 (plasmid w/ AG43) 

- 2mL LB broth 

- 2µL ampicillin

20/08/09 - Plasmid Digestion

19/08/09 - Miniprepping and Running PCR Gel + Nanodrop

18/08/09 - PCR Preparation and Picking Cultures

24/07/09 - Extraction of DNA from E.coli M5427

17/07/09 - MG1655 Stock Preparation
MG1655 E. coli grown on pure LB stock. No growth was observed using LB + ampicillin medium.
Pelleted and resuspended in 50:50 glycerol:LB medium and stored at -80 degrees C.
--->

Bioprecipitation Project

25/09/09 - Digestion of EL (mutant)

[...]

A gel was run with all 4 samples + ladder

Well Labels

• Well 1 = 1kb ladder
• Well 2 = Control EL (before mutagenesis)
• Well 3 = Control EL (before mutagenesis) + BamH1/Xba1
• Well 4 = Mutant EL
• Well 5 = Mutant EL + BamH1/Xba1

Click HERE for a picture of the Gel.

24/09/09 - Phosphorylation, Mutagenesis, Ligation for K (double mutant)

24/09/09 - DNA Miniprep of EL (mutant)

22/09/09 - Digestion of Miniprep (K - double mutant)

22/09/09 - Colony Pick
One colony was found for the EL transformation. This was picked and incubates at 37'C

21/09/09 - Nutrient Broth, Miniprep and Transformation
It was a busy day,

• Nutrient Broth was made. 13g/L of Nutrient Broth were made (Total of 4L)
• Miniprep (K - double mutant):
• Transformation (K - single mutants):

18/09/09 - Mutagenesis for PXCK-K and PXCK-EL
We have done mutagenesis on two of the plasmids. Here is a quick overview on the process: CLICK HERE

15/09/09 - Transformation with PXCK-K and PXCK-EL
PXCK-K and PXCK-EL plasmids were transformed into electrocompetent E. coli and left overnight at 37C in ampicillin plates (PXCK-K) or chloramphenicol plates (PXCK-EL).

08/09/09 Our Primers have arrived today, this means we can start to put our four genes into the standard plasmids!

02/09/09 For the last couple of weeks we have been trying to design primers in order to put them into the iGEM standard plasmids. Since restriction sites are against us, we are using mutagenesis in order to bypass this problem. Primers were ordered TODAY!

19/08/09 Miniprep of bacterial cultures. For procedure click HERE. Plasmids were stored in -20'C freezer (FIONA).

18/08/09 Colonies were picked from transformed bacteria. Protocol can be found by clicking HERE.

14/08/09
Results from transformation 2

• pxCK-EJ = 1 colony
• pxCK-ES = 3 colonies
• pxCK-EL = 27*4 colonies
• pxCK-K = 53*4 colonies

Plates were sealed with parafilm and stored in the -4 fridge.

13/08/09 A second transformation was performed on the four plasmids. Procedure slightly changed, click HERE to see it.

11/08/09 No results were given from the transformation. Transformation will have to be repeated.

10/08/09 Transformation of our 4 plasmids took place. They were transformed into TOP10 bacteria. A colony check will be preformed tomorrow morning.

To see protocol, click HERE

7/08/09 Vectors were examined and we are planning for the best cloning strategy for the standard plasmids.

6/08/09 Plasmid solutions were left overnight and in the morning checked again with Nanodrop. No DNA was measured. Samples were put in the waterbath at 37 degrees and left for a couple of hours. If Nanodrop does not work after the waterbath we will still do transformation since we only need few plasmids for the transformation to work. Nanodrop may not have been able to quantify such a small concentration of plasmids.

LB media was poured on petri dishes and plasmid DNA was checked again using Nanodrop.

• pXCK-EL = 10.55 ng/uL
• pXCK-ES = 8.43 ng/uL
• pXCK-K = 11.73 ng/uL
• pXCK-E/J = 2.66 ng/uL

Plasmids were stored and transformation will be done on Monday.

5/08/09

Plasmids have arrived!

• pXCK-E/J
• pXCK-K
• pXCK-EL
• pXCK-ES

We have put them into 150 mL of TE buffer and left overnight for the paper to dissolve and later extract the plasmid from the solution.

A Nanodrop was used to see if the DNA was coming out of the paper. No DNA was detected, thus samples were left overnight.

Click on the plasmid name to look at the vector