Team:PKU Beijing/Project/AND Gate 1/AND Gate Result

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Project

AND Gate 1 Result

Project > AND Gate 1 > AND Gate Result

This AND gate family is the earliest construction by us, and it made us much more confident about other AND Gates which were under construction at that time. There are nine AND gates in this family, and they are distinguished by the RBS of the T7ptag gene, while sharing the same promoters to drive T7ptag and SupD. They are , , , , , , , , and . And these AND Gates are on the plasmid backbone pSB4K5, which is low copy (about 5 copies) plasmid thus it facilitates further assembling.

In order to test the function of these AND gates, Shan Shen constructed a reporter GFP under the control of a T7 promoter, which will be activated if the T7 polymerase is successfully translated.(). And the reporter is on the plasmid .

The two plasmids are sequentially transformed to E.coli, a gradient of HSL and a gradient of IPTG is combined to test the behavior of the AND Gate under different combination of HSL and IPTG concentration. The result is read by a microplate reader. Data are processed and ploted using Matlab. (Fig 1, 2, 3 and 4. For more detail, refer to our protocol)

Fig1. GFP expression of AND gate, K228125: lux-SupD-lac-T7ptag (rbs: J44001). The data are showed for (left to right): 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 1×10^-4 and 1×10^-3M IPTG, and (bottom to top) 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 and 1×10^-4M HSL.

Fig2. GFP expression of AND gate, K228124: lux-SupD-lac-T7ptag (rbs: J61127). The data are showed for (left to right): 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 1×10^-4 and 1×10^-3M IPTG, and (bottom to top) 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 and 1×10^-4M HSL.

Fig3. GFP expression of AND gate, K228120: lux-SupD-lac-T7ptag (rbs: B0032). The data are showed for (left to right): 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 1×10^-4 and 1×10^-3M IPTG, and (bottom to top) 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 and 1×10^-4M HSL.

Fig4. GFP expression of AND gate, K228126 : lux-SupD-lac-T7ptag (rbs: B0033). The data are showed for (left to right): 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 1×10^-4 and 1×10^-3M IPTG, and (bottom to top) 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 and 1×10^-4M HSL.

Discussion:

From the result of some of these AND gates, we found that most of these AND Gates are always inducible by IPTG, but independent of the concentration of HSL. It indicates that the leakage of HSL sensor is significant, and the basal expression of SupD is so high that without HSL there is sufficient SupD to work on T7ptag. This result is consistent with the induction curve of HSL sensor.(there are quite amount of expression without HSL induction.)for more details, refer to Sensor Result.

Possible improvements: Because the leakage is on the promoter which drives SupD, we can not modulate the RBS (there is no RBS on SupD side) to make a qualified AND gate. If the leakage is on the T7ptag side, we may get some good results after modulate its RBS (change RBS or mutate it). The best solution is to replace the HSL sensor with other better promoters. Actually we got some better results by doing this. They are Given below.

The result of other five AND gates are not shown here.

AND Gates of Shuke Wu

This AND Gate family is made of pLac-supD & pBad-T7ptag.

The typical pattern of this AND gate is similar to Shan Shen's AND gates.

This family is the AND gate family constructed also the quickliest: at the same time with Shan Shen's AND gates. This AND gates family contents nine AND gates with the difference on the RBS of the T7ptag: K228823, K228824, K228825, K228826, K228827, K228828, K228829, K228830, and K228831. And these gates are on the plasmid pSB4K5.

In order to test the function of these AND gates, Shuke use a reporter GFP under the control of a T7 promoter: K228099. And the reporter is on the plasmid J61002.

Using the E.coli with the reporter plasmid, he transformed the AND gate plasmids into it, and tested them using various combinations of different concentration of IPTG and arabinose. The result is tested by a microplate reader (Fig 1, 2, 3 and 4. For more detail, refer to our protocol)

Fig1. GFP expression of AND gate, K228829: lac-SupD-pBad-T7ptag(rbs: B0033). The data are showed for (left to right): 1×10^-9, 1×10^-7, 1×10^-6, 1×10^-5 1×10^-4 and 1×10^-3M IPTG, and (bottom to top) 1×10^-9, 1×10^-7, 1×10^-6, 1×10^-5, 1×10^-4 and 1×10^-3M arabinose.

Fig2. GFP expression of AND gate, K228827: lac-SupD-pBad-T7ptag(rbs: B0030). The data are showed for (left to right): 1×10^-9, 1×10^-7, 1×10^-6, 1×10^-5 1×10^-4 and 1×10^-3M IPTG, and (bottom to top) 1×10^-9, 1×10^-7, 1×10^-6, 1×10^-5, 1×10^-4 and 1×10^-3M arabinose.

Fig3. GFP expression of AND gate, K228823: lac-SupD-pBad-T7ptag(rbs: J44001). The data are showed for (left to right): 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5 1×10^-4, 1×10^-3 and 1×10^-2M IPTG, and (bottom to top) 1×10^-9, 1×10^-8, 1×10^-7, 1×10^-6, 1×10^-5, 1×10^-4, 1×10^-3 and 1×10^-2M arabinose.

Fig4. GFP expression of AND gate, K228823: simplified version of Fig3. The data are showed for 1×10^-9 and 1×10^-2M IPTG, and 1×10^-9 and 1×10^-2M arabinose. The concentration of 1×10^-9 equals input=0, and 1×10^-2 equals input=1. So the four columns show the response of (0,0), (0,1), (1,0) and (1,1).

Discussion: AND gate K228829 (Fig 1) and K228823 (Fig 3) are the two best AND gates in this family, while others did not display AND gate behavior (Fig 2). It is a common sense that the activity range of promoters affects the function of the AND gate circuit (Anderson JC, Voigt CA, Arkin AP (2007) Environmental signal integration by a modular AND gate. Mol Syst Biol 3: 133). Thus we used nine different RBSs as a method to modulate the expression level of T7ptag and got some quite good AND gates (Fig 1&3). However, this strategy can not change the transcription of SupD, which may lead to leakage of AND gates. Because Shuke used lac system, which suffers from some problems (refer to our sensor result), to drive the SupD, this AND gate family can not have some perfect AND gates.

To improve, we should choose a good promoter, with little leakage, to drive SupD.

The result of other six AND gates are not shown here.

AND Gate of Guosheng Zhang (The Best AND Gate we achieve)

Other AND Gates

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@@ Line 11: / Line 11: @@
 <br>
 This AND gate family is the earliest construction by us, and it made us much more confident about other AND Gates which were under construction at that time.
-This AND Gate family contains nine AND gates distinguished by the RBS of the T7ptag gene. They are <partinfo>BBa_K228119</partinfo>, <partinfo>BBa_K228120</partinfo>, <partinfo>BBa_K228121</partinfo>, <partinfo>BBa_K228122</partinfo>, <partinfo>BBa_K228123</partinfo>, <partinfo>BBa_K228124</partinfo>, <partinfo>BBa_K228125</partinfo>, <partinfo>BBa_K228126</partinfo>, and <partinfo>BBa_K228127</partinfo>. And these AND Gates are on the plasmid pSB4K5.
+There are nine AND gates in this family, and they are distinguished by the RBS of the T7ptag gene, while sharing the same promoters to drive T7ptag and SupD. They are <partinfo>BBa_K228119</partinfo>, <partinfo>BBa_K228120</partinfo>, <partinfo>BBa_K228121</partinfo>, <partinfo>BBa_K228122</partinfo>, <partinfo>BBa_K228123</partinfo>, <partinfo>BBa_K228124</partinfo>, <partinfo>BBa_K228125</partinfo>, <partinfo>BBa_K228126</partinfo>, and <partinfo>BBa_K228127</partinfo>. And these AND Gates are on the plasmid backbone pSB4K5, which is low copy (about 5 copies) plasmid thus it facilitates further assembling.
-In order to test the function of these AND gates, Shan Shen constructed a reporter GFP under the control of a T7 promoter.(<partinfo>BBa_K228099</partinfo>). And the reporter is on the plasmid <partinfo>BBa_J61002</partinfo>.
+In order to test the function of these AND gates, Shan Shen constructed a reporter GFP under the control of a T7 promoter, which will be activated if the T7 polymerase is successfully translated.(<partinfo>BBa_K228099</partinfo>). And the reporter is on the plasmid <partinfo>BBa_J61002</partinfo>.
 The two plasmids are sequentially transformed to ''E.coli'', a gradient of HSL and a gradient of IPTG is combined to test the behavior of the AND Gate under different combination of HSL and IPTG concentration. The result is read by a microplate reader. Data are processed and ploted using Matlab. (Fig 1, 2, 3 and 4. For more detail, refer to our [[Team:PKU_Beijing/Notebook/Protocol/Chemical_Inducible_GFP|protocol]])
@@ Line 28: / Line 28: @@
 Discussion:
-From the result of some of these AND gates, we found that most of these AND Gates are inducible by IPTG, but independent of the concentration of HSL. It indicates that the leakage of HSL sensor is significant, and the basal expression of SupD is so high that without HSL there is sufficient SupD to work on T7ptag. This result is consistent with the induction curve of HSL sensor.(there are quite amount of expression without HSL induction.)for more details, refer to [[Team:PKU_Beijing/Project/AND_Gate_1/Inducible_System_Result|Sensor Result]].
+From the result of some of these AND gates, we found that most of these AND Gates are always inducible by IPTG, but independent of the concentration of HSL. It indicates that the leakage of HSL sensor is significant, and the basal expression of SupD is so high that without HSL there is sufficient SupD to work on T7ptag. This result is consistent with the induction curve of HSL sensor.(there are quite amount of expression without HSL induction.)for more details, refer to [[Team:PKU_Beijing/Project/AND_Gate_1/Inducible_System_Result|Sensor Result]].
 Possible improvements:
-Because the leakage is on the SupD side, we can not modulate the RBS (there is no RBS on SupD side) to make a qualified AND gate. If the leakage is on the T7ptag side, we may get some good results after modulate its RBS (change RBS or mutate it). The best solution is to replace the HSL sensor with other better promoters. Actually we got some better results by doing this. They are Given below.
+Because the leakage is on the promoter which drives SupD, we can not modulate the RBS (there is no RBS on SupD side) to make a qualified AND gate. If the leakage is on the T7ptag side, we may get some good results after modulate its RBS (change RBS or mutate it). The best solution is to replace the HSL sensor with other better promoters. Actually we got some better results by doing this. They are Given below.
 The result of other five AND gates are not shown here.