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From 2010.igem.org

Week-9

Identification of Bio-bricks

Plasmid of the samples which showed right size band on the gel were sent to a plasmid concentration test. We decided only F2, F6, F7, F8, F15 and F16 were in good concentration to proceed.

Sample	A[260]	A[280]	Bg[320]	Ratio	Protein (μg/ml)	Nucleic Acid (μg/ml)
f2	0,0946	0,0607	0,0202
f3	0,0444	0,0346	0,0216	1,7474	2,9830	0,9640
f6	0,0844	0,0669	0,0300	1,4721	16,1731	2,0934
f7	0,1348	0,1020	0,0557	1,7074	11,9965	3,3075
f8	0,0905	0,0690	0,0420	1,7961	5,1758	2,0767
f11	0,0332	0,0261	0,0184	1,9261	0,7189	0,6559
f15	0,0769	0,0511	0,0205	1,8439	4,7573	2,4456
f16	0,0543	0,0389	0,0223	1,9290	1,5147	1,4182

As the first PCR didn't show the length of all biobricks, the samples failed last time were sent to run another c-PCR. The result was showed in Fig.1

However, only sample F11 shows the band with right size on the gel pic. Samples, F1, F3, F4, F5, F9, F10, F11, F12, F13, F14 and F17 were inoculated again to get enough plasmid for further use. Second colony PCR was performed again for all the starting biobricks and the products were run for gel again. Comparing the DNA length to the band size, all the biobricks were confirmed, except for F16.

The concentration of plasmid were measured for all the samples and values were showed in Table 2

Analysis

Collection time 2010-08-05 14:34:11

   Sample        A[260]     A[280]    Bg[320]     Ratio     Protein   Nucleic Acid 

                                                             µg/ml       µg/ml     

____________________________________________________________________________________

f1 0,0183 0,0101 -0,0027 1,6428 3,9441 86,2531

f2 0,0477 0,0334 0,0160 1,8282 2,9083 137,1389

f3 0,0255 0,0132 -0,0021 1,8045 2,8374 118,5919

f4 0,0518 0,0398 0,0216 1,6573 5,4159 124,4569

f5 0,0108 0,0033 -0,0041 2,0017 0,2684 66,7742

f6 0,0137 0,0079 0,0006 1,7979 1,3839 56,0149

f7 0,0072 0,0010 -0,0071 1,7638 1,7529 60,7466

f8 -0,0035 -0,0080 -0,0151 1,6180 2,3362 47,0326

f9 0,0104 0,0035 -0,0054 1,7835 1,7833 67,4654

f10 0,0074 -0,0004 -0,0076 2,0846 -0,1927 68,6519

f11 0,0184 0,0088 -0,0063 1,6384 4,6986 101,2263

f12 0,0008 -0,0044 -0,0115 1,7349 1,6832 51,6802

f13 0,0157 0,0066 -0,0074 1,6499 4,2257 94,6587

f14 0,0033 -0,0031 -0,0100 1,9215 0,6691 58,6173

f15 0,0403 0,0345 0,0294 2,1530 -0,3966 50,2377

16 0,0182 0,0075 -0,0052 1,8335 2,0872 101,2592

f17 0,0082 0,0036 -0,0020 1,8332 0,9140 44,2838

Concentration before dilution for dection

ng/µL

f1 52

f2 82

f3 71

f4 75

f5 40

f6 34

f7 36

f8 28

f9 40

f10 41

f11 61

f12 31

f13 57

f14 35

f15 30

f16 61

F17 27

Plasmid of F1, F2, F4, F9,F10,F12, F17,ccdb C3 and ccdb K3 were digested with proper enzymes bought from Sigma-Aldrich. The biobricks were ligated in pairs according to the plan by Quick Ligase kit from Fermentas. The ligation product were transformed into DH5α competent cells and each sample were plated on the agar plate which antibiotics matched to its backbone antibiotic resistance.

Construction of G1- G7

5 colonies were picked from each of those plates and sent to perform c-PCR. According to the band size on gel picture, 1 or 2 colonies of each set were selected to inoculate.

Again, inoculated samples were sent to perform plasmid extraction and measure the plasmid concentration.

Table . Plasmid concentration of G sets and F16.

Plasmids were digested with proper enzymes bought from Sigma-Aldrich and run for gel to confirm the size.

Figure . Gel picture shows the length of digested G sets.

From the gel, we think G1 and G2C4 seems having the right biobricks in size. G5 seems not digested well, though the plasmid seems in right size. The colonies we picked for G3, G4, G6 seems in wrong size according to c-PCR. Another transformation is necessary for G3, G4, and G6 and the starting biobricks need to be checked again by gel.

Figure . Gel result to verify length of G5C3 and the starting material of G3, G4, and G6

All the starting material of G3, G4, and G6 seems good, though G5 C3 doesn’t seems in the right length. So a second transformation was done for G3, G4, G5, G6. After transformation, the cells were plated on the proper antibiotic plates. 12 colonies were picked from each plates and sent to perform c-PCR. The c-PCR result was shown on the gel picture.

Figure Gel picture shows the c-PCR result of second time transformation product of G3. The samples marked in orange were thought in right size and proceed to inoculate. Figure Gel picture shows the c-PCR result of second time transformation product of G4. The samples marked in orange were thought in right size and proceed to inoculate. Figure Gel picture shows the c-PCR result of second time transformation product of G5. The samples marked in orange were thought in right size and proceed to inoculate. Figure Gel picture shows the c-PCR result of second time transformation product of G6. The samples marked in orange were thought in right size and proceed to inoculate.

Selected colonies were inoculated and performed plasmid extraction on the second day. The plasmid concentration was measured and result is showed in Table 4

Table . Plasmid concentration of G3, G4, G6 sets

For further confirm the construction of G sets, we decided to use some specific digestion and run the gel to verify the special sites on each construct. The digestion enzymes were chosen by using Gentle.

Table . Specific enzymes chosen for each construct and the expected length of all digested parts Figure . Gel picture shows the result of specific digestion of G3, G4, G5 and G6.

Only samples from G4 and G6 showed in the expected length and consistence. A second specific digestion were performed to samples of G3 and G5

Figure . Gel picture shows the result of specifc digestion of G3 and G5

Comparing the expected length of digested parts, G3C5, G4C6, G5C12 and G6C6 were digested and ligated according to the plan