SBB09Ntbk-Sadao Ota

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Sadao 14:02, 6 May 2009 (EDT)

Summary of Activities up to now:
* The absorbance readings were inconclusive again - so trying to grow more cells and incubate more precipitates with arabinose
To do this week:
* grow more cells and incubate to mineralize more silver particles with arabinose
* Find the absorption wavelengths for the incubated cells
* take out supernatants, getting rid of pellets
* Find the absorption wavelengths for the dissoving ag particles, and Quantify the third round of results for the AG4 assay

before Wednesday

More incubations. TBS (4/24). fresh AgNO3. varied amount of cells and total volume of solution.

constructs 11-14 (1ml and 2ml) = 0.1mM AgNO3 in 200ul
constructs 15-17 and control (1ml) = 0.1mM AgNO3 in 1000ul
constructs 15-17 and control (2ml) = 1.0mM AgNO3 in 1000ul

incubate for 24 hours

Wednesday (for the grown cells):

results:


  • 2ml control, construct 15, construct 16, construct 17
  • Today, the color appeared in solution, not in precipitate form.
  • (the lack of color from constructs 11-14 might be because of small volume since previous experiment had shown color.)

Image:050609results table.jpg



control = series 1, construct15 = series 2, construct16 = series 3, construct17 = series 4

  • no noticeable peaks or differences between the constructs. perhaps scattering signal is too high because of the presence of cells.


Take out the supernatents, getting rid of background of cells

  • To get rid of the scattering signal, cells were spun down, and data was generated using the respective supernatants.


  • pelleted 2ml control, construct 15, construct 16, construct 17



control = series 1, construct15 = series 2, construct16 = series 3, construct17 = series 4

  • noticeable increased absorbance around 440nm-500nm.


Sadao 14:02, 29 April 2009 (EDT)

Summary of Activities up to now:
* The absorbance readings were inconclusive - so trying again (under light?)
To do today:
* Find the absorption wavelengths for the AG4 assay
* Quantify the second round of results for the AG4 assay

grew cells in 4ml LB and 4ul arabinose. used DH10B as control instead of 1363. Constructs are as below.

Results from incubation on Monday

control 1363 - white

  • 11 - white {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
  • 12 - reddish* {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
  • 13 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
  • 14 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
  • 15 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
  • 16 - white {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
  • 17 - reddish {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}


All of the tubes had reddish precipitate except the control. Construct 13 had the most precipitate and 16 had the least.

quantification

  • tried to resuspend the reddish particles, but did not resuspend despite lots of vortexing and other attempts.
  • tried to - 1. add lysozyme 2. manually remove precipitate and put into water 3. lots of vortexing
  • Took absorbance measurements of control, 16 (lysozyme), 15 (in water), and 13 (vortexing).

After normalizing the graph, the control had least absorbance, but there is no significant difference in signal in the 400-440nm range, so inconclusive results.

Sadao 14:02, 27 April 2009 (EDT)

Summary of Activities up to now:
* Over the weekend, we got the TBS+AgNO3 assays yielding reddish color for samples 12, 13, 14, 15
* Prepared a repeat of TBS+AgNO3 assay, and left overnight - labelled AG4 Assay V2
* The absorbance readings were inconclusive - the samples have too high background (due to the large presence of E.coli cells)
To do today:
* Prepare a second round of TBS+AgNO3 assay, under light
* Find the absorption wavelengths for the AG4 assay
* Quantify the first round of results for the AG4 assay

AG4 Silver biomineralization assay

Preparation of AgNO3 solution

  • Weigh AgNO3 powder in an Eppendorf tube (weighed 0.088 grams)
  • MW of AgNO3 is 169.87 - dilute 0.088g with 0.500ml offers ~1 M solution
  • Make 200ul of 1mM solutions by adding H2O

Preparation of cells for assay

  • Spin down 2ml of cells and throw away supernatant
  • Add 200 ul of TBS and resuspend cells
  • Spin down the cells and throw away supernatant

Running the assay on cells

  • Add 180 ul of TBS to each pellet (prepared above)
  • Resuspend cells
  • Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube
  • Incubate in the locker overnight at room temperature, according to the paper (ACS Nano, Kitae Nam et. al.)

Determining the AG4 absorption wavelengths

In order to get quantitative data, we need to find out which wavelengths are absorbed by the AG4 peptide silver precipitate. We chose a sample with good colour, and scanned it through the colour spectrum. The instrument used was the Tecan spectrophotometer, and measurements were taken. parameters: 250-800nm range. 10nm intervals. 96 well plate.

  • Results were inconclusive
  • Control and samples had very similar absorption spectra
  • Results probably due to presence of cells

we can get rid of the cells, but need to incubate cells again before that.

Sadao 13:20, 22 April 2009 (EDT)

Summary of Activities:
To do today:
==== Preparation of Tris buffer solution ====
* Add 605 mg Tris and 876 mg NaCl to 80 ml H2O
* Adjust pH to 7.4 by adding HCl and bring volume to 100 ml

==== Preparation of AgNO3 solution ====
* Weigh AgNO3 powder in an Eppendorf tube (weighed 0.013 grams)
* AgNO3 has MW 169.87 g/mol - dilute 0.013g with 0.750ml for ~10 mM solution
* Make 3ml of 1mM solutions by adding 100ul of 10mM AgNO3 into 900ul of H2O

==== Preparation of cells for assay ====
* Spin down 2ml of cells and throw away supernatant
* Add 200 ul of TBS and resuspend cells
* Spin down the cells and throw away supernatant

==== Preparing cells for growth in LBTAg media ====
* Dispense 3ml of LB media into 8 chambers in growth rack
* Add 200 ul of Tris buffer solution to the LB media
* Add 32 ul of 10 mM AgNO3 to make 0.1 mM LBTAg media
* Pick cells from each washed pellet (above) and add to corresponding growth chambers
* Cover the rack and incubate overnight at 37C

AG4 Silver biomineralization assay

Preparation of Tris buffer (1*) solution

adjust Ph 7.4 with adding 1M HCl

Preparation of AgNO3 solution

  • Weigh AgNO3 powder in an Eppendorf tube (weighed 0.013 grams)
  • AgNO3 has MW 169.87 g/mol - dilute 0.013g with 0.750ml for ~10 mM solution
  • Make 3ml of 1mM solutions by adding 100ul of 10mM AgNO3 into 900ul of H2O

Sadao 5:04, 21 March 2009 (PST)

I was not in lab today, but Susan and Roger carried out the following work:

Summary of Activities:
* Grew one colony from each sample (11-17) and control in 4ml LB media (with CA)
* Grew one colony from each sample (11-17) and control in 4ml LB media and 0.1mM AgNO3 (with CA)
* [http://openwetware.org/wiki/Image:Silver_binding1.doc growing cells grid]


Sadao 13:41, 20 April 2009 (EDT)

The composite parts have been assembled, and now we are doing assays for the different constructs. I am doing the AG4 silver binding peptide assay (Construct 18) with Carlos, Roger, Dirk, and Susan.

Our assay protocol can be found here:

Silver biomineralization assay

Summary of Activities:
* Tested 1M, 100mM, 10mM, 1mM, 0.5mM, 0.1mM AgNO3 in LB media
* A precipitate formed with AgNO3 in LB all the way down to 0.5mM
* 0.1mM AgNO3 in LB media did not form a visible precipitate
* Transformed constructs into bacteria (Roger and Susan)
* Plated the cells (Roger and Susan)
* We will use constructs 11-17 and a pBca9145-Bca1363 as control
To do today:
* Test AgNO3 precipitation in LB Media


AG4 Silver biomineralization assay

Preparation of AgNO3 solutions

  • Weigh AgNO3 powder in an Eppendorf tube (weighed 0.88 grams)
  • AgNO3 has MW 169.87 g/mol - dilute 0.88g with 0.5ml for ~10 M solution
  • Dilute it down to 10mM
  • Create 1 mM, 0.1 mM, and 0.05 mM dilutions by adding 9+1, 9+1, and 1+1 parts of H2O and AgNO3 solution

Test AgNO3 precipitation in LB media

  • Prepare Eppendorf tubes with 200ul of LB media
  • Dilute 1M, 100mM, 10mM, 1mM, 0.5mM, and 0.1mM AgNO3 in LB media
  • Mix and check for precipitate - if no precipitate, spin down in centrifuge and look for pellet

Sadao Ota 15:03, 9 February 2009 (EST)

My project is SBB09_20154, to make ag43 Display (short) ag43 Display (long) systems. Designed a oligo M10034 to make Autotransporter protein with promotors and so on (corresponding to amino acids 708-1017). Also, designed another oligo M10035 to make Autotransporter protein only (corresponding to amino acids 517-1017).


Sadao Ota 15:03, 16 February 2009 (EST)

Do PCR with the combination of Oso001&Oso002, and Oso002&Oso003.

PCR Cloning


Sadao Ota 15:03, 23 February 2009 (EST)

Clean up PCR products for gelation analysis
Clean up PCR products, Digest, Clean up and gel again.

Regular Zymo Cleanup (removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction) here

Load to Gel to perform gelation analysis (dead or alive) here

EcoRI/BamHI Digest of PCR Products (my PCR products are large enough, so no need to modificate the process) here

Sadao Ota 15:03, 25 February 2009 (EST)

Ligation of EcoRI/BamHI digests


I used gel to separate the DNA I need I also used digested DNA without gelation to do heat shock.

2nd PCR cloning did not work. So, PCRing again...

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