Part:BBa_K741012

plac-anticro-T -pCon(0.856)-RBS-crogfp-T

The fusion protein crogfp(λ phage Cro protein and GFP) is constantly expressed. The antisense RNA of cro is produced to inhibit the translation of the mRNA of crogfp after induced by IPTG.This part is used to measure the function of anticro.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 124
  Illegal BglII site found at 1481
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 918
  

To test the function of antisense RNA to cro (anticro) we have constructed, we measure the fluorescence intensity of plac-anticroT-pcon(0.856)-crogfp and plac-cI-prm-anticroT-pcon(0.856)-crogfp.

At the beginning, the unit fluorescence intensity of the experimental groups with glucose added is the lowest, because glucose promotes the growth of E.coli., which makes the rate of fluorescence divided by OD lower than the other two groups.

About 45 minutes later, the unit fluorescence intensity of experimental groups with glucose added is higher than the blank group, because glucose represses the expression of plac, which reduces the transcription of anticro. Therefore, the translation of crogfp is repressed less and the unit fluorescence intensity is higher.

On the contrast, IPTG activates plac, which increases the transcription of anticro. Anticro binds to the mRNA crogfp and represses its translation.

Data

plac-RBS-anticro-T-pCon(0.856)-crogfp-T
fluorescence intensity
t/min	Parallel	0	200mM glucose	400mM glucose	600mM glucose	800mM glucose	1000mM glucose	1000mM IPTG	M9 medium
0	1	739.054	752.571	737.122	749.3	697.779	733.053	750.115	11.844
	2	838.872	764.425	750.931	766.918	739.893	770.521	721.767
	3	752.342	778.138	764.078	780.378	757.977	747.533	772.383
45	1	1121.065	1127.129	1132.739	1143.486	1104.684	1131.022	1070.284	12.897
	2	1033.489	1079.895	1116.582	1157.266	1140.391	1144.527	1035.693
	3	1040.658	1084.647	1158.246	1187.787	1190.048	1225.269	1027.305
90	1	1309.754	1503.889	1436.975	1510.864	1499.763	1546.764	1143.771	12.698
	2	1290.674	1456.243	1444.75	1578.095	1411.988	1495.092	1101.698
	3	1415.663	1317.712701	1542.16481	1490.563687	1490.115468	1510.510219	1064.565494
135	1	1294.433	1417.68	1502.504	1581.499	1520.589	1573.465	1263.79	12.945
	2	1246.153	1466.956	1515.73	1548.78	1587.538	1559.81	1219.769
	3	1305.599	1453.067	1460.94	1553.158	971.939	1603.972	1271.341
180	1	1438.159	1592.059	1595.049	1599.125	1573.181	1445.849	1206.695	11.793
	2	1374.769	1432.318	1578.138	1643.468	1620.898	1525.023	1194.993
	3	1311.343	1556.093	1551.538	1507.415	1458.318	1539.911	1135.83
plac-RBS-anticro-T-pCon(0.856)-crogfp-T
OD
t/min	Parallel	0	200mM glucose	400mM glucose	600mM glucose	800mM glucose	1000mM glucose	1000mM IPTG	M9 medium
0	1	0.1052	0.1091	0.1117	0.1087	0.114	0.1052	0.1043	0.0374
	2	0.108	0.1124	0.1179	0.1161	0.1107	0.1109	0.1036
	3	0.1084	0.1991	0.1076	0.1124	0.112	0.1101	0.1006
45	1	0.1721	0.1657	0.1646	0.1601	0.1671	0.1805	0.1615	0.0412
	2	0.161	0.1697	0.1717	0.1705	0.162	0.1633	0.1586
	3	0.1593	0.1698	0.1633	0.1664	0.1676	0.1679	0.1518
90	1	0.2552	0.2604	0.2576	0.2532	0.2615	0.2659	0.2261	0.0379
	2	0.2502	0.2569	0.2674	0.2752	0.2548	0.2672	0.2276
	3	0.2545	0.2617	0.2587	0.2552	0.2694	0.2645	0.2176
135	1	0.2987	0.3379	0.3383	0.2969	0.3182	0.3331	0.2864	0.04
	2	0.3163	0.3263	0.3316	0.3306	0.3245	0.3233	0.3154
	3	0.3221	0.3347	0.3284	0.3196	0.3382	0.3358	0.3037
180	1	0.4115	0.412	0.4272	0.3793	0.4092	0.3986	0.3565	0.0406
	2	0.4051	0.3951	0.411	0.421	0.3996	0.3746	0.374
	3	0.4147	0.4107	0.3976	0.38	0.4029	0.3994	0.378