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From 2011.igem.org

The experiment of screening defective bacteria

Step-I—Screen by streak cultivation

        We get 10 single colonies of HB101 by streaking. And they’re labeled for No.1—No.10 and separately streak cultured. So on the square plates, we get ten parts which are separately culturing ten bacteria colonies. Then we prepare essential culture medium and complementary culture medium to culture them and use BL21 as the contrast to screen the defective bacteria. HB101 can’t synthetize Proline and thiamine. So we have to add these two elements.

Results in theory:

Real results:

        We can preliminarily screen No.1—No.6 regions of HB101 defective bacteria. The colonies of HB101 and BL21 are both small and almost transparent. We suspect it’s because medium’s formula are not so reasonable. We prepare to do a gradient of Thi’s concentration and use liquid mediume to replace solid medium to detect.

MM medium

Medium optimization experiment Bacteria in right tube was DH5α for contrast

Step-II Medium optimization

         We choose colonies in No.1 region and culture them in liquid medium which have the formula of Thi’s concentration. 12h later, we measured OD of test tubes. BL21 have turn turbid，but HB101 not. We kept culturing HB101, but we thought we are fail at first. In order to analyze the failure reason, we culture DH5α(detective in Pro and Thi but not Leu) in the same liquid medium. If DH5αgrow well, it can be proved that the leucine added doesn’t work.
However, a total 35h later, HB101 turn turbid, which can prove the detective bacteria designed by us can work well. Then we measure OD to choose the most private concentration of Thi.

        By contrasting, we can get the best concentration of Thi for bacteria’s growth.
        ①: 10mg/100ml ②: 1mg/ml ③: 0.1mg/100ml ④: 0.01mg/100ml
        Results: HB101 in No.1 region are available. The best concentration of Thi is 1mg/100ml.

Basic information of defective bacteria and the medium’s formula

The genotype of strain—HB101: supE44,△(mcrc-mrr),recA13,ara-14,proA2,lacY1,galK2, rpsL20, xyL-5, mrl-1,leuB6, rhi-1.
M 9 basic medium
Make up 1L medium, 750ml aseptic and deionized water(refrigerated to 50℃ or under 50 ℃) should be added:
（1）5*M9 salt solution 200ml
        Use aseptic and deionized water solute these salts and make the volume to reach 1L:
        Na2HPO4·7H2O 64g
        KH2PO4 15g
        NaCl 2.5g
        NH4Cl 5.0g
        Subdivide 200ml as a share and steam sterilize at 15psi(1.05kg/cm2)
(2) 1mol/L MgSO4 2ml
(3) 20% solution of suitable carbon source ( like 20% glucose)
(4) 1mol/L CaCl2
         If necessary, we can add proper amino acid and vitamin to M9 medium.
         Make up MgSO4 and CaCl2 solution separately and autoclave them. Use aseptic water to make 5*M9 salt solution diluted to 980ml and then add MgSO4 ，CaCl2 and glucose solution to it. Use 0.22mm filter membrane to eliminate bacteria.