Team:TU Delft/8 June 2010 content

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Pieter with eppy

Contents

Eppy

Pieter just got a little present from Mariska van Ham :-) He's so happy with is eppy!

Today Nadine & Luke are learning how to work with HTML.

Lab work

BioBrick stocks

A number of Biobricks from the DNA distribution plates were suspended yesterday. Hugo and Kira did a transformation with 2 μL of the following BioBricks (antibiotic):

  • pSB3T5 (Tetracycline)
  • pSB1T3 (Tetracycline)
  • pSB1C3 (Chloramphenicol)
  • pSB3C5 (Chloramphenicol)
  • pSB1K3 (Kanamycin)
  • pSB1A3 (Ampicillin)
  • I13401 (Ampicillin)

Because our own competent cells were not good enough we used commercially available competent Top10 cells (Invitrogen). The cells were plated on LB-agar with the appropriate antibiotic. Biobricks plated We will have to wait until tomorrow to see if they were successful or not. Hopefully we will see some colonies tomorrow. Wish us luck!

Thias PCRed some BioBricks with various primer concentrations and at different temperatures to see which PCR method would work best. The PCR products were tested on a gel, and we saw a difference between the primer concentrations and the temperatures used.

Media and Solutions

By Hugo

Nadine and I are planing to revive our Ps. putida strain (muahaha). So, we prepared some solid media for that purpose: LB agar and E2 agar.

For LB medium this is the recipe that we use:

  • Tryptone 10g
  • Yeast extract 5 g
  • NaCl 10 g
  • Agar 15 g
  • Water, as much as you need for 1 L of culture medium

For E2 medium this is the recipe that we use:

  • (NH4)2HPO4 10 g
  • K2HPO4 5 g
  • Na2SO4 0.5g
  • 1 ml of MT stock solution
  • 1 ml of 1M MgSO4
  • Water, as much as you need for 1 L of culture medium

If you want to prepare the solid medium, add 15 g of agar per liter of medium.

This medium is really minimal, you can choose your own carbon source. Some literature recommend to use citrate, glucose, glycerol and, for our case, hydrocarbons. E2 medium is not complete without the following solution:

MT stock solution

  • FeSO4 7H2O 2.8 g
  • MnCl2 0.19g
  • CoCl2 6H2O 2.8 g
  • CaCl2 4H2O 1.84g
  • ZnSO4 7H2O 0.28 g
  • CuSO4 0.16g

WARNING: Dear iGEM people be aware that the original E2 composition is the following (Lageveen et al., 1988):

  • KH2PO4 3.7 g
  • K2HPO4 7.5 g
  • NaNH4HPO4.4H2O 3.5 g
  • Citrate 5 g / 12 g
  • 1 M MgSO4 . 7H2O 1 ml
  • MT stock solution 1 ml

and the original MT stock solution composition is:

  • FeSO4.7H2O 2.8 g
  • MnSO4.H2O 2 g
  • CoCl2.6H2O 2.8 g
  • CaCl2.2H2O 1.48 g
  • ZnSO4.7H2O 0.28 g
  • CuCl2.2H2O 0.16 g

Why did we change the medium?

First, we lack of the weird salt NaNH4HPO4.4H2O, we want to save some money by making some engineering in the culture medium. Instead of the main salts for E2 we are using the salts recommended by [http://www.lgcstandards-atcc.org/ American Type Culture Collection] (ATCC) in the optimal medium for Ps. putida, this mineral medium is also known as P1. Find the original medium composition [http://www.lgcstandards-atcc.org/Attachments/4091.pdf here].

Second, we decided to keep the MT stock solution because, alkB2 (one of our biobricks) requires certain minerals. Without this cofactors the protein won't work, however we lack of CuCl2 and MnSO4. So, we changed those salts for CuSO4 and MnCl2, we kept the molarity of the ions Cu+2 and Mn+2 (we made calculations and everything!). With these changes we hope to save some money and keep the richness of the medium intact (ish).

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