Difference between revisions of "Part:BBa K2797013"

Latest revision as of 22:22, 17 October 2018

High copy BioBrick assembly plasmid pSB1C3 with RFP internal standard

pSB1C3 with a constitutively expressed RFP (Red Fluorescent Protein) construct under the control of a medium strength Anderson promoter.

Usage and Biology

Newcastle iGEM 2018

In summer 2018, [http://2018.igem.org/Team:Newcastle/InterLab Team Newcastle] designed a constitutive, medium strength RFP expression construct. This construct was cloned into the backbone of the iGEM InterLab test device pSB1C3 vectors - in the non-coding region between the chloramphenicol resistance gene and the Origin of Replication - for use as an Internal Standard within the iGEM 2018 InterLab study. This part is an improvement on the pSB1C3 plasmid.

The addition of this RFP Internal Standard within the InterLab study devices revealed that, even under the same experimental conditions, the complex nature of biological systems still cause variation in protein expression, and highlights how important the use of an Internal Standard is in the identification of variation in part characterisation studies.

Major Findings

Data from the RFP Internal Standard experiments is shown in figure 1, and from this data the following conclusions were gained.

Lower target gene expression - Fluorescein/OD regarding GFPmut3b was lower in pSB1C3 modified with the RFP Internal Standard construct with reference to those without.
Target gene expression consistency - The presence of the Internal Standard in pSB1C3, while causes target gene (GFPmut3b) expression to be lower, allows the target gene expression to be more consistent with little to no fluctuations in expression over 24 hours in respect to pSB1C3 target gene expression without the Internal Standard construct.
Transcription/translation machinery saturation - The stronger promoters (Test Devices 1 and 4) showed little to no fluorescence on the basis of RFP, despite sequencing revealing the presence of the gene. It is thought that these promoters are so strong that the addition of any other protein expression genes cause the saturation of transcription/translation mechanisms
Variation in protein expression - In devices were RFP fluorescence can be observed, there is significant variation in the expression of the gene despite the same experimental conditions, indicating that protein expression may be more complex than originally first thought.

Figure 1. A) Scatter plots showing the relationship between Relative AFUs (GFPmut3b fluorescein/OD) against time (x-axis) for the original test device and the internal standard test devices. The legend for each device is shown on the left of each graph. Over 24 hours, the original test devices reach peak fluorescence at around the 6-7 hour mark, with devices 4 & 1 being the strongest and 3 & 6 being the weakest. After the 6 hour mark there is a decrease in fluorescence to the 22 hour mark where fluorescence beings to increase once again. The internal standard devices all showed a consistent increase with no decrease over 24 hours. Device 2 was the most fluorescent with devices 1 and 4 being significantly lower than the original group. Fluorescence was lower for each device initially than the original but eventually surpassed the original study as the fluorescence decreased over time. B) Scatter plots showing the relationship between Relative AFUs (RFP fluorescence/OD) (y-axis) and time (x-axis) for the RFP internal standard test devices. The legend for each device is shown on the left of the graph. Over the 24 period, the control groups and devices 1 & 4 exhibited a fluorescence/OD of 0. The remaining other devices - 2, 3 & 6 – all showed a consistent positive increase in fluorescence/OD. Device 2 however has the highest fluorescence/OD throughout the study, followed by devices 6, 3 and 2 with fluorescence/OD values of 0.064, 0.048 and 0.038 respectively. These groups began fluorescing between 200 and 440 min.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found in sequence at 989
Illegal suffix found in sequence at 1
Illegal EcoRI site found at 3052
Illegal XbaI site found at 3067
Illegal SpeI site found at 1905
Illegal PstI site found at 1919
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 989
Illegal EcoRI site found at 3052
Illegal NheI site found at 1017
Illegal NheI site found at 1040
Illegal SpeI site found at 2
Illegal SpeI site found at 1905
Illegal PstI site found at 16
Illegal PstI site found at 1919
Illegal NotI site found at 9
Illegal NotI site found at 995
Illegal NotI site found at 1912
Illegal NotI site found at 3058
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 989
Illegal EcoRI site found at 3052
Illegal XhoI site found at 2036
Illegal XhoI site found at 2928
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found in sequence at 989
Illegal suffix found in sequence at 2
Illegal EcoRI site found at 3052
Illegal XbaI site found at 3067
Illegal SpeI site found at 1905
Illegal PstI site found at 1919
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found in sequence at 989
Illegal EcoRI site found at 3052
Illegal XbaI site found at 1004
Illegal XbaI site found at 3067
Illegal SpeI site found at 2
Illegal SpeI site found at 1905
Illegal PstI site found at 16
Illegal PstI site found at 1919
Illegal AgeI site found at 1627
Illegal AgeI site found at 1739
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2797013 short</partinfo>
-pSB1C3 with a constituitively expressed RFP construct under the control of a medium strength Anderson promoter.
+pSB1C3 with a constitutively expressed RFP (Red Fluorescent Protein) construct under the control of a medium strength Anderson promoter.
 ===Usage and Biology===
@@ Line 9: / Line 9: @@
 '''Newcastle iGEM 2018'''
-In summer 2018, [http://2018.igem.org/Team:Newcastle/InterLab Newcastle iGEM GeneMachine] designed a constitutive, medium strength RFP expression construct and cloned said construct into the backbone of the iGEM InterLab test device pSB1C3 vectors - in the non-coding region between the chloramphenicol resistance gene and the Origin of Replication - for use as an Internal Standard within the iGEM 2018 InterLab study.
+In summer 2018, [http://2018.igem.org/Team:Newcastle/InterLab Team Newcastle] designed a constitutive, medium strength RFP expression construct. This construct was cloned into the backbone of the iGEM InterLab test device pSB1C3 vectors - in the non-coding region between the chloramphenicol resistance gene and the Origin of Replication - for use as an Internal Standard within the iGEM 2018 InterLab study. This part is an improvement on the [[Part:pSB1C3|pSB1C3]] plasmid.
 The addition of this RFP Internal Standard within the InterLab study devices revealed that, even under the same experimental conditions, the complex nature of biological systems still cause variation in protein expression, and highlights how important the use of an Internal Standard is in the identification of variation in part characterisation studies.
-'''Major Findings '''
+'''Major Findings'''
-*'''Lower target gene expression''' - Fluorescein/OD regarding GFPmut3b was lower in pSB1C3 modified with the RFP Internal Standard construct with reference to those without (Figure 1).
+Data from the RFP Internal Standard experiments is shown in figure 1, and from this data the following conclusions were gained.
-*'''Target gene expression consistency''' - The presence of the Internal Standard in pSB1C3, while causes target gene (GFPmut3b) expression to be lower, allows the target gene expression to be more consistent with little to no fluctuations in expression over 24 hours in respect to pSB1C3 target gene expression without the Internal Standard construct (Figure 1).
-*'''Transcription/translation machinery saturation''' - The stronger promoters (Test Devices 1 and 4) showed little to no fluorescence on the basis of RFP, despite sequencing revealing the presence of the gene. It is thought that these promoters are so strong that the addition of any other protein expression genes cause the saturation of transcription/translation mechanisms (Figure 2)
-*'''Variation in protein expression''' - In devices were RFP fluorescence can be observed, there is significant variation in the expression of the gene despite the same experimental conditions, indicating that protein expression may be more complex than originally first thought (Figure 2).
+*'''Lower target gene expression''' - Fluorescein/OD regarding GFPmut3b was lower in pSB1C3 modified with the RFP Internal Standard construct with reference to those without.
+*'''Target gene expression consistency''' - The presence of the Internal Standard in pSB1C3, while causes target gene (GFPmut3b) expression to be lower, allows the target gene expression to be more consistent with little to no fluctuations in expression over 24 hours in respect to pSB1C3 target gene expression without the Internal Standard construct.
+*'''Transcription/translation machinery saturation''' - The stronger promoters (Test Devices 1 and 4) showed little to no fluorescence on the basis of RFP, despite sequencing revealing the presence of the gene. It is thought that these promoters are so strong that the addition of any other protein expression genes cause the saturation of transcription/translation mechanisms
+*'''Variation in protein expression''' - In devices were RFP fluorescence can be observed, there is significant variation in the expression of the gene despite the same experimental conditions, indicating that protein expression may be more complex than originally first thought.
-[[Image:RFPGFP.png|thumb|center|500px|'''Figure 1. Scatter plots showing the relationship between µm fluorescein/OD (GFPmut3b fluorescence) against time for the original test devices (above) and the internal standard test devices (below).''' The y-axis shows µm fluorescein/OD and the x-axis shows time in minutes. On each plot, the legend for each device is shown at the top of the graph. Over 24 hours, the original test devices reach peak fluorescence at around the 6-7 hour mark, with devices 4 & 1 being the strongest and 3 & 6 being the weakest. After the 6 hour mark there is a decrease in fluorescence to the 22 hour mark where fluorescence beings to increase once again. The internal standard devices all showed a consistent increase with no decrease over 24 hours. Device 2 was the most fluorescent with devices 1 and 4 being significantly lower than the original group. Fluorescence was lower for each device initially than the original but eventually surpassed the original study as the fluorescence decreased over time.]]
-[[Image: RFPRFP.png|thumb|center|500px| '''Figure 2. Scatter plots showing the relationship between fluorescence/OD (RFP fluorescence) and time for the RFP internal standard test devices.''' The y-axis shows fluorescence/OD, while the x-axis shows time in minutes. The legend for each device is shown at the top of the graph. Over the 24 period, the control groups and devices 1 & 4 exhibited a fluorescence/OD of 0. The remaining other devices - 2, 3, 5 & 6 – all showed a consistent positive increase in fluorescence/OD. Device 2 however has the highest fluorescence/OD throughout the study, followed by devices 6, 3 and 2 with fluorescence/OD values of 0.064, 0.048 and 0.038 respectively. These groups began fluorescing between 200 and 440 mins.]]
+[[Image:IntS.png|thumb|center|500px|'''Figure 1. A) Scatter plots showing the relationship between Relative AFUs (GFPmut3b fluorescein/OD) against time (x-axis) for the original test device and the internal standard test devices.''' The legend for each device is shown on the left of each graph. Over 24 hours, the original test devices reach peak fluorescence at around the 6-7 hour mark, with devices 4 & 1 being the strongest and 3 & 6 being the weakest. After the 6 hour mark there is a decrease in fluorescence to the 22 hour mark where fluorescence beings to increase once again. The internal standard devices all showed a consistent increase with no decrease over 24 hours. Device 2 was the most fluorescent with devices 1 and 4 being significantly lower than the original group. Fluorescence was lower for each device initially than the original but eventually surpassed the original study as the fluorescence decreased over time.
+'''B) Scatter plots showing the relationship between Relative AFUs (RFP fluorescence/OD) (y-axis) and time (x-axis) for the RFP internal standard test devices.''' The legend for each device is shown on the left of the graph. Over the 24 period, the control groups and devices 1 & 4 exhibited a fluorescence/OD of 0. The remaining other devices - 2, 3 & 6 – all showed a consistent positive increase in fluorescence/OD. Device 2 however has the highest fluorescence/OD throughout the study, followed by devices 6, 3 and 2 with fluorescence/OD values of 0.064, 0.048 and 0.038 respectively. These groups began fluorescing between 200 and 440 min.
+]]
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