BUGSS:Notebook/Lead Sensor

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Project Description/Abstract

The BUGSS team has been working to cultivate a strain of E. Coli that produces metallothionein and RFP in the presence of heavy metals like lead. Adequate production of metallothionein protein can sequester lead in order to make the metal non-bioavailable. Again, the BUGSS team is building on earlier work on lead sequestration,utilizing some parts from the iGEM registry while producing and submitting new and composite parts. Sequences were found and edited for metallothionein production (BmtA), and the PbrR regulator. PpbrA promoters were connected to RFP and Bmta genes in order to regulate metallothienein and RFP production, inducing their generation in the presence of lead. In order for this new construct to work, the E. Coli's natural heavy metal resistance gene, Znta, must be deactivated. The team is utilizing a one step gene inactivation protocol using phage lambda red recombinase to recombine the gene with a kanamycin resistance insert.

Protocols

iGem Plasmid Backbone Protocols
Addgene Transofrmation Protocols

Daily Notes

March 20, 2015

Master Mix Had Results but others didn't. Ran the following control reactions:

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March 18, 2015

Gel run on March 14th suggested that the reactions had too much template. Ran the following controls with less template:



Image:3_18_Table.JPG

Plan for next week: PCR of znta knockout using pkd20 linear plasmid.


March 13, 2015

Performed colony PCR As Follows:

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March 4, 2015

Ran gel on colony PCR. Results inconclusive. Lacked a positive control.


February 27, 2015

  • Experimental colony only yielded 2 colonies.

Performed Colony PCR on Part 69 Transformants

Image:2_27_Table.JPG




February 18, 2015

Performed transformation protocol found on open wetware using 20μL in 1mL SOC. Cells used were DH5α.

Each tube used 5μL of DNA in 100μL of cells.
1. Part 69 Ligation
2. No Insert Control
3. No Ligase Control
4. PSB1C3 only positive control.
5. Negative control with cells only.

To do next: Check transformants and colony PCR screening.

February 11, 2015

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Image:2_11_Table.JPG

To do: Transformations!

February 4, 2015
Digested cut vector(Red) and Made uncut(Blue) vector control. Image:2_04Table.JPG


February 2, 2015
Ran gel on digested and EcoR1 digested PSB1C3 samples. Used 10μL sample/2μL loading dye in each and compared to 1kb ladder.
(Used 2 trials on each with different loading dye to make sure the dye worked.):

Bands on the digested lane seem to be around 1.4kb. Strongest intensity bands on undigested lane seem to be somewhat smaller than 4kb.
Image:WIN_20150202_210307_(2).JPG

Filename: PSB1C3_Verifications_2_02_15 (in Maurice folder)




January 28, 2015

Past: #71 – verified by PCR for size. We have vector with #71
Let's focus on #69 – then, if cells have both #71 and 69 we'll get fluorescence in presence of zinc.

Vector PSB1C3 25ug/ml (green 1.5ml eppendorf, IGEM box)
is this our vector?
Cut it with EcoRI => linear. Run gel with cut and uncut.
Restriction reaction set up by Maurice:

Image:1_28Table.JPG

1h at 37C. Then frozen in Maurice's box (-20C).

To do: Verify PSB1C3 with electrophoresis. Ligate 3 remaining parts cut with Xba1 and Spe1.

December, 2014

PCR done with Positive (F2620 1299bp) and Negative Control (Template DNA)

DNA 2μL
VF2 1.3μL
VR 1.3μL
dNTPs 1.0μL
Taq Buffer 5.0μL
Taq Enzyme 1.0μL
Water 38.1μL


November 20, 2014

Performed plasmid miniprep on cmr transformant. A260: 0.072 = 3.6Ξμg/mL A260/280=1.6
Plasmid is in green tube in IGEM box marked 71 PSB1C3 on top
Put 1mL + 1mL glycerol stock in -70°C BUGSS BOX
Remainder placed in fridge with (RED) pSB1C3 + J23039 & control plasmid

Next step: Digest & Run Gel on plasmid
Do PCR with VF2/VR primers to verify insert.


November 12, 2014
Used part 71, a negative transformation control and a PSB13 positive control in a transformation using the Addgene protocol. (http://www.addgene.org/plasmid-protocols/bacterial-transformation/)

Used 3 plates of commercial competent cells and 3 plates of lab-prepared (from CAT119) competent cells.

Plates labeled:
Factory cells:

71 Supercomp
P Supercomp
N Supercomp

Lab cells:

71 Norm
P Norm
N Norm

To do:
Make more competent cells and chloramphenicol plates.
Make liquid culture.
Make zinc plates.
Grow test cultures of transformations in the presence of zinc and pcr assay for znta knockout.

November 5, 2014
Previously digested vector and parts with SpeI and XbaI
Used digested vector and Ligated 4 parts each into separate, linear pSB1C3 vectors:

BB-RBS-BmtA-tt-BB, Labeled 68
BB-PpbrA-RBS-RFP-tt-BB, Labeled 69
BB-PpbrA-RBS-Bmta-ttt-BB, Labeled 70
BB-J23101-RBS-PbrR-tttt-BB, Labeled 71


And one positive (P with RFP) and negative (N) control.

All reactions left overnight.

Using igem protocol: http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones

To do: Grow cells with PSB1C3 positive control and cut out RFP fragment by digestion and gel isolation.
Tuesday(Jesse and crew): start a culture of CAT119. Wednesday (crew): dilute and grow culture and possibly do transformation.

[11/5 Tables and Record]

Usage:

2.5 μL T4 Ligase
6 μL T4 Buffer

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