iGEM 2015 — Team UNC-Chapel_Hill

# Team:UNC-Chapel Hill/Notebook

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* [NOTEBOOK](/Team%3AUNC-Chapel_Hill/Notebook)
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|  | NOTEBOOK | | |  |

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|  | [May](#May) | [June](#June) | [July](#July) | [August](#August) | [September](#September) |  |

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|  | Notebook Format  Month Day, Year Name(s) of people who completed work Protocol(s) used More detailed description May 18th, 2015  Connor and Sean Transformation Resuspended parts BBa\_E1010 and BBa\_I716120 on plate 3 well 11n and plate 3 well 11m, respectively. Transformed both and placed in 37 degree incubator \*Note: parts left in 37 degree incubator too long (>24 hours) and overgrew May 21st, 2015  May 21st, 2015 Elliot and Danny Transformation Resuspended part BBa\_K118011 on plate 3 well 20c. Transformed this part along with BBa\_E1010 again.   June 3rd, 2015  Connor and Sean Transformation Resuspended parts BBa\_K1033916 and BBa\_K592009 on plate 4 well 6m and plate 1 well 15e, respectively. Transformed those parts and BBa\_E1010. \*Note: BBa\_K1033916 plate was thrown out due to suspicion of LBKanR mislabeling and the two other chromoproteins appear to have been switched. Elliot and Danny Transformation Transformed parts BBa\_E1010, BBa\_K1033916, BBa\_K592009, BBa\_K861171 and BBa\_K118011. Will and Jerry ZR Plasmid Miniprep Classic Miniprepped parts BBa\_K861171 and BBa\_K118011. Prepared 1x TAE Buffer from stock. June 4th, 2015  Will Transformation Did overall inventory and plated BBa\_K861171 Connor and Sean Liquid Cultures Created liquid cultures of BBa\_E1010 (2), BBa\_K592009 (2), and BBa\_K1033916 and placed in 37 degree shaking incubator overnight. \*Note: Liquid cultures spent >24 hours in 37 degree incubator, so thrown out. Also, BBa\_K118011 failed to grow. June 5th, 2015  Elliot and Danny Liquid Cultures Created liquid cultures of BBa\_E1010 (2), BBa\_K592009 (2), and BBa\_K1033916 and placed in 37 degree shaking incubator overnight. LB was autoclaved and placed in 4 degree fridge. June 6th, 2015  Elliot and Danny ZR Plasmid Miniprep Classic Miniprepped BBa\_E1010 (2), BBa\_K592009 (2), and BBa\_K1033916 and nanodropped for concentrations. June 8th, 2015  Will Did overall inventory and made a 1L DI water stock June 10th, 2015  Will and Jerry PCR Received and reconstituted MLC Parts and MLC primers. Performed PCR amplification on the MLC parts. June 11th, 2015  Will and Jerry Agarose Gel Electrophoresis, DNA Clean + Concentrator Prep, PCR Ran a gel verifying the MLC parts, concentrated the PCR-generated plasmids and nanodropped. After determining that concentrations were insufficient, ran another PCR for MLC amplification. Also conducted inventory. Connor and Sean DNA Clean + Concentrator Prep, Transformation Prepped the DNA and transformed BBa\_J4450, which was reconstituted from plate 4 well 4b. Made a glycerol stock. Elliot and Danny Transformation Created ampicillin plates and reconstituted BBa\_J61100 on plate 4 well b4. Transformed BBa\_J61100 on the ampicillin plates. June 12th, 2015  Connor, Sean and Elliot Liquid Cultures, DNA Clean and Concentrator Prep Made 2 liquid cultures of BBa\_J61100 (LBAmp) and 4 liquid cultures of BBa\_J4450 (LBCam). Prepped the new MLC PCR mixes and nanodropped for concentrations. June 13th, 2015  Elliot and Danny ZR Plasmid Miniprep Classic Miniprepped the liquid cultures of BBa\_J61100 and BBa\_J4450 (6 in total). Stored in the -20 degrees freezer. June 14th, 2015  Elliot and Danny Liquid Cultures Created 3 liquid cultures of BBa\_J4450 (LBCam) and 6 liquid cultures of BBa\_J61100 (LBAmp). June 15th, 2015  Connor ZR Plasmid Miniprep Classic Miniprepped 2 BBa\_J4450 cultures and 5 BBa\_J61100 cultures. Nanodropped and stored in the -20 degrees freezer. Elliot and Danny 3A Assembly, Agarose Gel Electrophoresis Conducted 3A Assembly with the following components: pSB1C3 (from BBa\_J4450), BBa\_K1033916/BBa\_E1010/BBa\_K592009 (downstream component) and BBa\_J61100 (upstream component). Made a 1% agarose gel with the following wells: 1: 1 kb ladder; 2: BBa\_E1010 #3; 3. BBa\_E1010 #2; 4. BBa\_K1033916 #3; 5. BBa\_K592009 #2; 6. BBa\_K861171 # 3; 7. 1 kb ladder. Made another 1% agarose gel: 1: 1 kb ladder; 2: 100 bp ladder; 3. BBa\_K1033916 #3; 4. BBa\_K592009 #2; 5. BBa\_K1033916 #1; 6. BBa\_K861171 #1; 7. BBa\_K861171 #2; 8. BBa\_K861171 #3 June 16th, 2015  Elliot + Danny + Jerry Agarose Gel Electrophoresis Ran a diagnostic 2% gel for the repressible. Will + Jerry Agarose Gel Electrophoresis Ran a 1% gel with the following specifications: 1: 1 kb ladder; 2: 100 bp ladder; 3: BBa\_K1033916 #1; 4: BBa\_K1033916 #2; 5: BBa\_K1033916 #3; 6: BBa\_K592009 #2; 7: BBa\_K861171 #1; 8: BBa\_K861171 #2; 9: BBa\_K861171 #3 Connor + Will 3A Assembly, Simple Recombination Conducted a 3A assembly with the following components: pSB1C3, BBa\_E1010/BBa\_K592009, BBa\_J1100. Conducted simple recombination (cutting a backbone and “part” with the same enzymes) with the following components: MLC1/2/3/4, pSB1C3. Will Transformation Transformed the two 3A plasmids and four simple recombination plasmids. \*Note: all MLC plates failed to grow June 17th, 2015  Will PCR, Transformation Conducted another PCR amplification procedure for the order MLC parts (1-4). Transformed the MLC + pSB1C3 constructs that failed to grow once more. June 18th, 2015  Will Agarose Gel Electrophoresis, Gel DNA Recovery Prep Made glycerol stocks of BBa\_K1033916#3/pSB1C3/BBa\_J61100 (pink, so actually BBa\_J4450), BBa\_K592009#2/pSB1C3/BBa\_J61100, BBa\_E1010#2/pSB1C3/BBa\_J61100. Then ran a 1% gel with the following specifications: 1: 1 kb ladder; 2: 100 bp ladder; 3: MLC1: 4: MLC2; 5: MLC3; 6: MLC4; 7: MasterMix; 8: USP H20. Then ran a DNA gel recovery prep and nanodropped. \*Note: MLC remainders from the gel were not enough, so another PCR is recommended. Connor Zyppy Plasmid Miniprep, Transformation Miniprepped BBa\_K592009/BBa\_J61100/pSB1C3 and BBa\_E1010/BBa\_J61100/pSB1C3 liquid cultures. Nanodropped the results. Transformed the BBa\_1033916 #1/#2 3A results. Jerry, Danny, Elliot 3A Assembly, Transformation, Agarose Gel Electrophoresis Conducted a 3A with the following components: BBa\_J4450 (pSB1C3), BBa\_J61100, BBa\_K1033916 #1/2. Ran a 1% digest gel with the following specifications: 1: 1 kb ladder; 2: BBa\_K1033916 #1, undigested; 3: BBa\_K1033916 #1, EcoRI digested; 4: BBa\_K1033916 #1, SpeI digested; 5: BBa\_K1033916 #1, double digest; 6: BBa\_K1033916 #2, undigested; 7: BBa\_K1033916 #2, EcoRI digested; 8: BBa\_K1033916 #2, SpeI digested; 9: BBa\_K1033916 #2, double digest; 10: BBa\_K861171 #1, undigested; 11: BBa\_K861171 #1, EcoRI digested; 12: BBa\_K861171 #1, SpeI digested; 13: BBa\_K861171 #1, double digest; 14: 100 bp ladder. June 19th, 2015  Connor, Sean PCR, DNA Clean + Concentration Prep, Simple Recombination, Liquid Culture Redid PCR amplification for MLC parts and prepped the PCR mix with the concentrator kit. Conducted the following simple recombination: pSB1C3 + MLC1/2/3/4. Made a liquid culture for the BBa\_K1033916/BBa\_J61100/pSB1C3 plasmid. June 20th, 2015  Sean Transformation Transformed the simple recombination results (MLC1-4 + backbone). June 21th, 2015  Sean Transformation, Liquid Cultures Retransformed the simple recombination results due to bad colony growth. Made liquid cultures for MLC1, MLC2 (#1 and #2) and MLC 3. Elliot Agarose Gel Electrophoresis Ran a 1% gel with the following specifications: 1: 1 kb ladder; 2: BBa\_E1010#1/BBa\_J61100/pSB1C3, undigested; 3: BBa\_E1010#1/BBa\_J61100/pSB1C3, EcoRI digestion; 4: BBa\_E1010#1/BBa\_J61100/pSB1C3, SpeI digestion; 5: BBa\_E1010#1/BBa\_J61100/pSB1C3, double digest; 6: BBa\_E1010#3/BBa\_J61100/pSB1C3, undigested; 7: BBa\_E1010#3/BBa\_J61100/pSB1C3, EcoRI digestion; 8: BBa\_E1010#3/BBa\_J61100/pSB1C3, SpeI digestion; 9: BBa\_E1010#3/BBa\_J61100/pSB1C3, double digest; 10: 1 kb ladder. June 22nd, 2015  Jerry Agarose Gel Electrophoresis Ran a 1% gel with the following specifications: 1: 100 bp ladder; 2: MLC1; 3: MLC2; 4: MLC3; 5: MLC4; 6: MLC2#1/BBa\_J61100/pSB1C3; 7: MLC2#2/BBa\_J61100/pSB1C3; 8: MLC3/BBa\_J61100/pSB1C3; 9: 1 kb ladder. June 23rd, 2015  Jerry 3A Assembly Conducted a 3A assembly with the following components: BBa\_J4450 (pSB1C3), BBa\_K1033916#2+BBa\_J61100/BBa\_K592009+BBa\_J61100, BBa\_K861171/ BBa\_K118011. Only managed to get to the digestion step, stored for Connors’ usage tomorrow. Connor Zyppy Plasmid Miniprep Miniprepped the MLC1/2(1)/2(2)/3+pSB1C3 liquid cultures. Made more LB stock. June 24th, 2015  Connor 3A Assembly, Zyppy Plasmid Miniprep Finished the ligation step of 3A assembly and stored the plasmids in the freezer. Made glycerol stocks for MLC 1/2/3 (1 is mislabeled as 4!). Miniprepped the MLC1+pSB1C3 construct and nanodropped for concentration determination. June 26th, 2015  Elliot ZR Plasmid Miniprep Classic Miniprepped 3 BBa\_K1033916/BBa\_J61100/pSB1C3 construct liquid cultures July 1st, 2015  Elliot Liquid Cultures, Transformation Made liquid cultures for the BBa\_1033916/BBa\_J61100/pSB1C3 and the BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 constructs. Also plated the BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 and BBa\_K861171/BBa\_1033916+BBa\_J61100/pSB1C3 constructs once more. Connor 3A Assembly Constructed a plasmid with the following components: BBa\_E1010+BBa\_J61100 + BBa\_K4450 + MLC1/2(1)/2(2)/3. July 2nd, 2015  Connor Transformation/Liquid Culture Transformed the 3A assembly results from yesterday (BBa\_E1010/BBa\_J61100 + BBa\_K4450 + MLC1/2(1)/2(2)/3). Also made liquid cultures of the BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 and BBa\_K861171/BBa\_1033916+BBa\_J61100/pSB1C3 constructs. \*NOTE: plates were overgrown during selection! July 3rd, 2015  Connor 3A Assembly/Transformation/Liquid Culture Attempted another ligation with the backbone (BBa\_J4450) and MLC4 digests. Transformed the result. Made liquid cultures of MLC composite parts (BBa\_E1010/BBa\_J61100 + BBa\_K4450 + MLC1/2(1)/2(2)/3). \*Note: The MLC4 ligation culture failed to grow. Elliot ZR Plasmid Miniprep Classic Miniprepped the BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 and BBa\_K861171/BBa\_1033916+BBa\_J61100/pSB1C3 constructs. Nanodropped and found no DNA, so discarded. \*Note: Liquid cultures had a cellular deposit on the bottom for some reason July 4th, 2015  Connor ZR Plasmid Miniprep Classic/Liquid Cultures Miniprepped the MLC Composite Parts (BBa\_E1010/BBa\_J61100 + BBa\_K4450 + MLC1/2(1)/2(2)/3). Nanodropped and stored the results. Made liquid cultures of the BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 and BBa\_K861171/BBa\_1033916+BBa\_J61100/pSB1C3 constructs July 6th, 2015  Connor Simple Recombination/Agarose Gel Electrophoresis/Transformation Attempted simple recombination with MLC4 and pSB1C3. Transformed the result. Ran a 1% gel with the following specifications: 1. 1 kb ladder; 2. 100 bp ladder; 3. MLC1/BBa\_E1010/pSB1C3 (E+P cut); 4. MLC2(1)/BBa\_E1010/pSB1C3 (E+P cut); 5. MLC2(2)/BBa\_E1010/pSB1C3 (E+P cut); 6. MLC3/BBa\_E1010/pSB1C3 (E+P cut). July 7th, 2015  Connor and Jerry ZR Plasmid Miniprep Classic/Agarose Gel Electrophoresis Miniprepped liquid cultures from the red and white colonie on the MLC Composite Parts plates and the BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 and BBa\_K861171/BBa\_1033916+BBa\_J61100/pSB1C3 constructs. Ran a 1% gel with the following specifications: 1. 1 kb ladder; 2. 100 bp ladder; 3. MLC1/BBa\_E1010/pSB1C3 (E+P cut) (Red); 4. MLC2(1)/BBa\_E1010/pSB1C3 (E+P cut) (Red); 5. MLC2(2)/BBa\_E1010/pSB1C (E+P cut) (Red)3; 6. BBa\_K861171/BBa\_1033916+BBa\_J61100/pSB1C3 (E+P cut); 7. BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 (E+P cut); 8. MLC1/BBa\_E1010/pSB1C3 (E+P cut) (White); 4. MLC2(1)/BBa\_E1010/pSB1C3 (E+P cut) (White); 5. MLC2(2)/BBa\_E1010/pSB1C (E+P cut) (White). July 8th, 2015  Connor Liquid Cultures Made liquid cultures of the BBa\_K118011/BBa\_K592009+BBa\_J61100/pSB1C3 and BBa\_K861171/BBa\_1033916+BBa\_J61100/pSB1C3 constructs. July 9th, 2015  Connor ZR Plasmid Miniprep Classic Miniprepped the BBa\_K118011, BBa\_K861171, BBa\_K592009, BBa\_K1033916, BBa\_E1010 and BBa\_E1010/BBa\_J61100 constructs and nanodropped. Made glycerol stocks of all. July 10th, 2015  Connor and Jerry Liquid Culture/Transformation Transformed BBa\_B0034 from plate 4, well 1N with 10 uL USP water. Transformed and plated with AmpR plates. Made 4 liquid cultures with 1g/L glucose concentration and the MLC Composite Parts to check for chromoprotein expression via glucose induction. July 11th, 2015  Connor Liquid Culture/Simple Recombination/Transformation Made liquid cultures of BBa\_B0034 and BBa\_J61100. Attempted simple recombination with MLC4(1)/MLC4(2) and pSB1C3. Transformed the ligation result. July 12th, 2015  Connor ZR Plasmid Miniprep Classic/Liquid Cultures Miniprepped the BBa\_J61100 and BBa\_B0034. Nanodropped and made a glycerol stock of both. Made liquid cultures of the MLC4(1)/pSB1C3 and MLC4(2)/pSB1C3 constructs. July 13th, 2015  Connor ZR Plasmid Miniprep Classic/3A Assembly/Agarose Gel Electrophoresis/Transformation Miniprepped the MLC4(1)/pSB1C3 and MLC4(2)/pSB1C3 constructs. Conducted 3A assembly to make the following parts: BBa\_K1033916/BBa\_B0034; BBa\_K1033916/BBa\_J61100; BBa\_K592009/BBa\_B0034; BBa\_K592009/BBa\_J61100; BBa\_E1010/BBa\_B0034; BBa\_E1010/BBa\_J61100. Transformed the ligation results. Ran a 1% gel with the following specifications: 1. 100 bp ladder; 2. 1 kb ladder; 3. MLC4(1)/pSB1C3; 4. MLC4(2)/pSB1C3; 5. pSB1C3(1); 6.pSB1C3(2). July 14th, 2015  Jerry PCR Amplified the MLC4 fragment July 15th, 2015  Elliot Transformation Plated BBa\_K1033931, BBa\_K1073023, and BBa\_K1033929. July 16th, 2015  Connor and Jerry DNA Clean and Concentrator Prep Prepped the MLC4 PCR results and nanodropped. Jerry Agarose Gel Electrophoresis/PCR Ran a 1% gel with the following specifications: 1. 100 bp ladder; 2. 1 kb ladder; 3. MLC4; 4. BBa\_K1033931; 5. BBa\_E1010. Amplified the MLC1-3 fragments with PCR. July 17th, 2015  Connor DNA Clean and Concentrator Prep/Agarose Gel Electrophoresis Prepped the MLC1-3 PCR Results and nanodropped. Ran a 1% gel with the following specifications: 1. 1 kb ladder; 2. 100 bp ladder; 3. MLC1; 4. MLC2; 5. MLC3; 6. MLC4. Jerry/Elliot Liquid Culture Made 2 liquid cultures of BBa\_K1073025, BBa\_K1073023 and BBa\_K1073021 each. Also made liquid cultures of each of the MLC+pSB1C3 constructs. July 18th, 2015  Elliot ZR Plasmid Miniprep Classic/3A Assembly Miniprepped BBa\_K1033931, BBa\_K1033929, and BBa\_K1073023. Nanodropped and created the following constructs with 3A: BBa\_K118011/BBa\_K1073023 and BBa\_K861171/BBa\_K1033929. Connor Transformation Transformed the following constructs: BBa\_K861171/BBa\_K1033929; BBa\_K118011/BBa\_K1073023; MLC1/pSB1C3; MLC2/pSB1C3; MLC3/pSB1C3; MLC4/pSB1C3. July 20th, 2015  Connor Liquid Culture/Agarose Gel Electrophoresis Made liquid cultures for each of the MLC Constructs in backbones. Ran a 1% gel with the following specifications: 1. 100 bp ladder; 2. 1 kb ladder; 3. BBa\_K118011/BBa\_K1073023 (1) (White); 4. BBa\_K118011/BBa\_K1073023 (2) (White); 5. BBa\_K118011/BBa\_K1073023 (1) (Red); 6. BBa\_K118011/BBa\_K1073023 (2) (Red); 7. BBa\_K861171/BBa\_K1033929 (1); 8. BBa\_K861171/BBa\_K1033929 (2). July 21st, 2015  Connor/Elliot Agarose Gel Electrophoresis Ran a 1% gel with the following specifications: 1. 1 kb ladder; 2. 100 bp ladder; 3. BBa\_K118011/BBa\_K1073023 (1) (White) (E+P cut); 4. BBa\_K118011/BBa\_K1073023 (2) (White) (E+P cut); 5. BBa\_K118011/BBa\_K1073023 (1) (Red) (E+P cut); 6. BBa\_K118011/BBa\_K1073023 (2) (Red) (E+P cut); 7. BBa\_K861171/BBa\_K1033929 (1) (E+P cut); 8. BBa\_K861171/BBa\_K1033929 (2) (E+P cut). July 23rd, 2015  Jerry Agarose Gel Electrophoresis Ran a 1% gel with the following specifications (all E+P cut): 1. 1 kb ladder; 2. 100 bp ladder; 3. MLC1/pSB1C3 (a); 4. MLC1/pSB1C3 (b); 5. MLC1/pSB1C3 (c); 6. MLC2/pSB1C3 (b); 7. MLC2/pSB1C3 (c); 8. MLC3/pSB1C3 (b); 9. MLC4/pSB1C3 (a); 10. MLC4/pSB1C3 (b); 11. BBa\_K861171/BBa\_K1033929; 12. BBa\_K118011/BBa\_K1073023; 13. 100 bp ladder; 14. 1 kb ladder. July 29th, 2015  Jerry Liquid Culture Made liquid cultures of BBa\_K118011 and BBa\_K861171. Also plated the BBa\_K1073023, BBa\_K1033929, and BBa\_K1033931 sent from the registry. July 30th, 2015 Jerry ZR Plasmid Miniprep Classic Miniprepped the BBa\_K118011 and BBa\_K861171 cultures. Nanodropped the result. July 31st, 2015  Jerry 3A Assembly/Simple Recombination/Agarose Gel Electrophoresis Conducted a 3A to create the following constructs: BBa\_K861171/BBa\_K1033929 and BBa\_K118011/BBa\_K1073023. Attempted to recombine the MLC fragments into the pSB1C3 backbone. Ran a 1% gel with the following  specifications: 1. 1 kb ladder; 2. 100 bp ladder; 3. pSB1C3 (E cut); 4. pSB1C3 (P cut); 5. pSB1C3; 6. MLC1 (E+S cut); 7. MLC2 (E+S cut); 8. MLC3 (E+S cut); 9. MLC4 (E+S cut); 10. pSB1C3 (E+S cut); 11. BBa\_K1073023 (P+X cut); 12. BBa\_K107029 (P+X cut); 13. BBa\_K861171 (P+S cut); 14. BBa\_K118011 (P+S cut). August 1st, 2015  Connor Transformation Transformed all the MLC/pSB1C3 constructs with one of the BBa\_K118011/BBa\_K1073023 (Red) and BBa\_K861171/BBa\_K1033929 constructs. August 2nd, 2015  Jerry Liquid Culture Made 2 liquid cultures each of the MLC constructs with pSB1C3, BBa\_K118011/BBa\_K1073023, and BBa\_K861171/BBa\_K1033929. August 3rd, 2015  Jerry Transformation Transformed the reconstituted BBa\_J23119 (plate 3, well 17) along with two of the BBa\_K118011/BBa\_K1073023 constructs and one of the BBa\_K861171/BBa\_K1033929 constructs. Jerry and Sean ZR Plasmid Miniprep Classic Prepped the MLC+pSB1C3, BBa\_K118011/BBa\_K1073023, and BBa\_K861171/BBa\_K1033929 constructs. Nanodropped the results. August 4th, 2015  Connor 3A Assembly/ZR Plasmid Miniprep Classic Attempted a 3A to make the following constructs: MLC1/BBa\_K1033931; MLC2/BBa\_K1033931; MLC3/BBa\_K1033931; MLC4/BBa\_K1033931. Miniprepped the pSB1C3 liquid cultures and nanodropped. Sean Elongated Digestion/DNA Clean and Concentrator Prep/Elongated Ligation Digested BBa\_J23119, BBa\_K1033931, BBa\_K1073023, and BBa\_K1033929 for 2 hours. Prepped the digestion results and nanodropped. Ligated and left overnight. Elliot Transformation Transformed MLC1-4+pSB1C3. August 5th, 2015  Connor/Elliot Liquid Culture/Transformation/Agarose Gel Electrophoresis Made 4 liquid cultures of the MLC/BBa\_K1033931 Constructs. Transformed the constitutive promoter (BBa\_J23119)+chromoprotein constructs. Ran a 1% gel with the following specifications: 1. 1 kb ladder; 2. 100 bp ladder; 3. BBa\_K118011/BBa\_K1073023 (1); 4. BBa\_K118011/BBa\_K1073023 (2); 5. BBa\_K861171/BBa\_K1033929 (1); 6. BBa\_K861171/BBa\_K1033929 (2). August 6th, 2015  Elliot ZR Plasmid Miniprep Classic Miniprepped the MLC1-4/pSB1C3/BBa\_K1033931 liquid cultures and nanodroppped. Sean DNA Clean and Concentrator Prep/Elongated Ligation Prepped the digestion results of BBa\_K861171, BBa\_K118011, BBa\_K1033929, and BBa\_K1073023. Ligated and left overnight. August 7th, 2015  Connor Transformation Transformed 2 uL of reconstituted pSB1A3 from plate 4, well 2H. August 9th, 2015  Connor Liquid Culture Made a liquid culture of pSB1A3. August 10th, 2015  Elliot Transformation Transformed 2 uL of reconstituted pSB1A3 from plate 4, well 2H. August 11th, 2015  Elliot Liquid Culture Made a liquid culture of pSB1A3. August 12th, 2015  Jerry/Sean Elongated Digestion/Agarose Gel Electrophoresis Digested MLC+pSB1C3, BBa\_K861171, and BBa\_K118011 at S+P. Digested BBa\_K1033929, BBa\_K1073023 and BBa\_K1033931 at X+P. Digested for 3 hours in total. Ran a 1% gel with the following specifications: 1. 1 kb ladder; 2. BBa\_K1073023; 3. BBa\_K1033929; 4. BBa\_K1033931; 5. BBa\_K118011; 6. BBa\_K861171; 7. MLC1+pSB1C3; 8. MLC2+pSB1C3; 9. MLC+pSB1C3; 10. BBa\_J23119. Sean Gel Purification Prep/Elongated Ligation Ran the digestion results on a gel and purified. Nanodropped and made ligation mixes for 3 hour incubation. Elliot Transformation Transformed the final ligation mixtures, including BBa\_K118011/BBa\_K1073023, BBa\_K861171/BBa\_K1033931, and MLC1-4/BBa\_K1033929. August 13th, 2015  Elliot Transformation Retransformed the final ligation mixtures, including BBa\_K118011/BBa\_K1073023, BBa\_K861171/BBa\_K1033931, and MLC1-4/BBa\_K1033929. August 14th, 2015  Jerry and Sean Elongated Digestion Digested MLC+pSB1C3, BBa\_K861171, and BBa\_K118011 at S+P. Digested BBa\_K1033929, BBa\_K1073023 and BBa\_K1033931 at X+P. Digested for 3 hours in total. August 18th, 2015  Sean DNA Clean and Concentrator/Gel Purification Prep/Elongated Ligation Prepped the promoter parts with the concentrator kit and selected for the correct chromoproteins with the gel purification prep. Ligated BBa\_J23119 and BBa\_K118011 with BBa\_K1073023 and left for 3 hours. Connor Transformation/Liquid Culture Transformed the red chromoprotein+constitutive promoter construct and the BBa\_K118011/BBa\_K1073023 construct. Made liquid cultures of all of the MLC/pSB1C3 constructs, BBa\_1033931, and BBa\_K1033929.  August 19th, 2015  Connor ZR Plasmid Miniprep Classic/Liquid Culture Miniprepped BBa\_J23119 and BBa\_K1073023. Made liquid cultures of BBa\_K118011, BBa\_K861171, BBa\_K1073023, BBa\_K1033931, BBa\_K1033929, the MLC1-4+pSB1C3 constructs and BBa\_J23119.  August 20th, 2015  Connor ZR Plasmid Miniprep Classic Miniprepped BBa\_K118011, BBa\_K861171, BBa\_K1073023, BBa\_K1033931, BBa\_K1033929, the MLC1-4+pSB1C3 constructs and BBa\_J23119 and nanodropped for concentration.  August 21st, 2015  Connor Elongated Digestion/Gel Purification Prep/Elongated Ligation Digested 2500 ng of BBa\_K118011, BBa\_K861171, BBa\_K1073023, BBa\_K1033931, BBa\_K1033929, the MLC1-4+pSB1C3 constructs and BBa\_J23119 each for 3 hours. Ran on a 1% gel and purified with the kit. Ligated overnight.  August 22nd, 2015  Connor Transformation Transformed the ligation mixes of BBa\_K118011, BBa\_K861171, BBa\_K1073023, BBa\_K1033931, BBa\_K1033929, the MLC1-4+pSB1C3 constructs and BBa\_J23119. Incubated overnight.  August 26th, 2015  Connor ZR Plasmid Miniprep Miniprepped BBa\_K118011/BBa\_K1073023, BBa\_K861171/BBa\_K1073023, and the MLC2 construct/BBa\_K1033931. Jerry PCR Amplified the PCR Constructs with a 32 cycle PCR.  August 27th, 2015  Sean PCR Amplified the PCR Constructs with a 35 cycle PCR and left overnight in the freezer. August 28th, 2015  Sean and Jerry DNA Clean and Concentrate Prep/Elongated Digestion Cleaned the completed PCR solutions with the prep. Nanodropped for concentrations. Digested the PCR constructs with pSB1C3 and incubated for three hours. Connor DNA Clean and Concentrator Prep/Ligation Prepped the digested PCR constructs and ligated into the digested pSB1C3 backbone. Incubated overnight.  August 29th, 2015  Connor Transformation Transformed the ligated PCR+pSB1C3 constructs. Incubated overnight.  August 30th, 2015  Elliot Transformation Transformed pSB1C3 stocks and plated. Incubated overnight.  August 31st, 2015  Connor PCR/DNA Clean and Concentrator Kit/Elongated Digestion/Elongated Ligation Conducted a 32 cycle PCR amplification of the PCR constructs. Cleaned with the kit and nanodropped. The results  were then digested with miniprepped pSB1C3 and cleaned again after a 2 hour incubation. The PCR constructs were then ligated with the digested pSB1C3 and incubated overnight.  September 1st, 2015  Jerry Transformation/PCR Transformed the PCR Construct ligation mixes and incubated overnight. Set a 32 cycle PCR to amplify more of the base PCR constructs.  September 2nd, 2015  Connor Liquid Culture Made liquid cultures of the MLC/BBa\_K1033931 PCR constructs, the BBa\_J23119/BBa\_K1073023 PCR construct, the BBa\_K861171/BBa\_K1073023 PCR construct, the BBa\_K118011/BBa\_K1033929 PCR construct and the tripart constructs.  September 3rd, 2015  Connor ZR Plasmid Miniprep Classic Miniprepped the MLC/BBa\_K1033931 PCR constructs, the BBa\_J23119/BBa\_K1073023 PCR construct, the BBa\_K861171/BBa\_K1073023 PCR construct, the BBa\_K118011/BBa\_K1033929 PCR construct and the tripart constructs.  September 4th, 2015  Jerry ZR Plasmid Miniprep Classic Miniprepped the viable PCR Construct liquid cultures and nanodropped. Connor PCR/DNA Clean and Concentrator Kit/Elongated DIgestion/Elongated Ligation Conducted a 32 cycle PCR amplification of the PCR constructs. Cleaned with the kit and nanodropped. The results were then digested with miniprepped pSB1C3 and cleaned again after a 2 hour incubation. The PCR constructs were then ligated with the digested pSB1C3 and incubated overnight.  September 5th, 2015  Will DNA Clean and Concentrator Prep/Elongated Ligation Prepped the BBa\_J23119/BBa\_K1033931 and BBa\_J23119/BBa\_K1033929 PCR constructs. Ligated with digested pSB1C3 and incubated overnight. Connor Transformation Transformed the ligated PCR constructs+pSB1C3. Incubated overnight.  September 6th, 2015  Elliot Liquid Cultures/Transformation Created liquid cultures of the viable PCR constructs and transformed the BBa\_J23119/BBa\_K1033931 and BBa\_J23119/BBa\_K1033929 PCR constructs. Incubated overnight.  September 7th, 2015  Elliot ZR Plasmid Miniprep Classic Miniprepped the liquid cultures of the viable PCR constructs.  September 8th, 2015  Connor PCR/Transformation Transformed the the ligated PCR constructs+pSB1C3. Incubated overnight. Also ran a 32 cycle PCR amplification of the BBa\_J23119/BBa\_K1033931 and BBa\_J23119/BBa\_K1033929 PCR constructs.  September 10th, 2015  Connor ZR Plasmid Miniprep Classic/PCR/Liquid Cultures/DNa Clean and Concentrator Prep Miniprepped the three MLC PCR Constructs. Amplified the BBa\_J23119/BBa\_K1033931, BBa\_J23119/BBa\_K1033929, BBa\_J23119/BBa\_K1073023, BBa\_K861171/BBa\_K1073023, MLC1/BBa\_K1033931, and Tripart PCR constructs with a 32 cycle PCR. Made liquid cultures of pSB1C3 and BBa\_B0034 and used the clean kit to prep the PCR products.  September 11th, 2015  Connor ZR Plasmid Miniprep Classic/Elongated Ligation/Elongated Digestion/Transformation Miniprepped the pSB1C3 and BBa\_B0034 cultures. Digested the PCR constructs, pSB1C3 and BBa\_B0034 stocks and incubated for two hours. Ligated the PCR constructs together with the backbones and incubated for 3 hours. Transformed and incubated overnight.  September 12th, 2015  Connor Liquid Cultures Prepared liquid cultures of the BBa\_J23119/BBa\_K1073023, BBa\_J23119/BBa\_K1033929, and BBa\_K118011/BBa\_K1033929 PCR constructs.  September 13th, 2015  Jerry Characterized the sensitivity parts by varying the amount of glucose concentrations in liquid cultures and monitoring their growth and changes in absorbance over time and with based on glucose concentrations. Connor ZR Plasmid Miniprep Classic Miniprepped pSB1C3 liquid cultures and the BBa\_K861171/BBa\_K1073023 stocks. Nanodropped and stored.  September 14th, 2015  Connor Elongated Digestion/Elongated Ligation/Transformation Digested three pSB1C3 stocks and one BBa\_B0034 stock along with 500 ng of purified PCR constructs. Incubated for three hours. Ligated together to insert the constructs into the backbone and incubated for 3 hours. Transformed and incubated overnight.  September 15th, 2015  Connor Liquid Culture Made liquid cultures of all of the viable PCR constructs, excluding the MLC1 construct and the full constructs. |

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