Team:Stony Brook/Notebook



Welcome to our notebook, the log of all our hard work this summer! Below are the summaries of what occurred each week; if you would like to see more detail, click on the weeks to expand it. 

 May 30-June 2 

Week 0

‣ Met with our advisors and worked on construct design for this week

‣ Set up the lab for start of wet lab work

June 3-9

Week 1

‣ Received our cyanobacteria and started culturing them

‣ Started a growth experiment to determine ideal growing conditions in our incubators (1 paper vs 3 sheets of paper)  

June 10-16

Week 2

‣ Continuation of growth experiments for cyanobacteria

‣ Spectrophotometry measurements for growth experiment

‣ Primers design for PCR and PCR information

‣ Spectrophotometric measurements to determine growth curve of cyanobacteria 

June 17-23

Week 3

‣ Continuation of spectrophotometry measurements for growth curve

‣ Started work for E. coli plasmid cultures by making agar plates

‣ Tested antibiotic solutions (streptomycin and spectinomycin) on plasmids

‣ Miniprep of 1579 DNA 

June 24-30

Week 4

‣ Continuation of spectrophotometry measurements

‣ Splitting/growing and culture of cyanobacteria

‣ Growing up plasmids received from Addgene

‣ Constructs from IDT arrived and primers for PCR arrived

‣ Both constructs and primers were resuspended

‣ PCR and PCR purification of all constructs that arrived

‣ Ran multiple gels on plasmid cultures and tested restriction enzymes in digest

‣ Start of Interlab Calibration 1/Calibration 2 

July 1-7 

Week 5

‣ Continuation of spectrophotometry measurements

‣ Splitting/growing and culture of cyanobacteria

‣ Gel extraction and gel purification for plasmids

‣ Interlab - Finished Calibration 1/2/3

‣ Made chloramphenicol stock

‣ PCR and gel purification of constructs  

July 8-14

Week 6

‣ PCR, gel extraction and gel purification of constructs

‣ Gel purification of plasmids

‣ Made glycerol stock of plasmids and frozen stocks of cyanobacteria

‣ Splitting/growing and culture of cyanobacteria

‣ Started Interlab transformation and inoculation of E. coli (Day 2) 

July 15-21

Week 7

‣ Splitting/growing and culture of cyanobacteria

‣ Finished interlab inoculation of E. coli colonies (Day 2)

‣ Gel extraction and purification of constructs (troubleshooting)

‣ PCR adjustments and trouble shooting for constructs

‣ Miniprep, RE digest and DNA purification of plasmids

‣ Starting work with CO2 incubator for growth of cyanobacteria

‣ Obtained light data on CO2 for the incubator 

July 22-28  

Week 8

‣ Splitting/growing and culture of cyanobacteria

‣ Interlab Day 3 - colony forming units protocol

‣ Contamination experiments for cyanobacteria

‣ PCR of constructs and ran products on gel

‣ First HiFi transformation of PCR product for cpc (with positive and negative controls)  

July 29-August 4 

Week 9

‣ Splitting/growing and culture of cyanobacteria

‣ PCR of constructs and running them on a gel

‣ PCR purification of products

‣ HiFi of cpc, cpc 560, lone cscB, idiA, optimized EYFP and original EYFP

‣ Inoculated colonies with HiFi

‣ Made BG-11 plates for culture of transformed cyanobacteria

‣ Transformation of cyanobacteria with lone cscB, cpc, cpc 560

‣ PCR troubleshooting

‣ Gel purification troubleshooting

‣ Sodium bicarbonate growth experiments  

August 5-11

Week 10

‣ Sodium bicarbonate growth experiments

‣ Gel purification troubleshooting

‣ Splitting/growing and culture of cyanobacteria

‣ Cyanobacteria transformation of rbc

‣ HiFi for idiA, EYFP and positive controls

‣ PCR troubleshooting  

August 12-18

Week 11

‣ Sodium bicarbonate growth experiments

‣ Splitting/growing and culture of cyanobacteria

‣ Transformed and plated HiFi for Opto EYFP and orig. EYFP

‣ Restreaked cyanobacteria plates

‣ Transformation of idiA, psba2, rbc

‣ Miniprep and inoculation for biobricks

‣ Start of sucrose assay experiments  

August 19-25

Week 12

‣ Sodium bicarbonate growth experiments

‣ Splitting/growing and culture of cyanobacteria

‣ Sucrose assay experiments

‣ Biobrick miniprep

‣ Colony PCR

‣ Transformation of cyanobacteria  

August 26-September 1

Week 13

‣ Cyanobacteria sucrose assays with cscB

‣ PCR and PCR purification for biobrick group

‣ Transformed cyanobacteria with psbA2 and plated transformed bacteria 

September 2-8 

Week 14

‣ Digestion of pSB1C3 and ligation with constructs

‣ Transformation of ligated cpc560

‣ Inoculation of cpc560, idiA

‣ Made BG-11 media for inoculations 

September 9-15 

Week 15

‣ Miniprep of transformed cpc560

‣ Inoculation of psbA2 colonies for cyanobacteria

‣ Ligation and Transformation of cpc for biobrick

‣ Luciferase assays for cpc

‣ Making LB and CAM plates for biobrick

‣ Inoculation of cpc

‣ Sucrose assay for cscB 

September 16-22  

Week 16

‣ Miniprep of idiA, cpc

‣ PCR of cscB, psbA2 and PCR purification

‣ Ligation and transformation of psbA2

‣ Another luciferase experiment  

September 23-29

Week 17

‣ Sequencing for biobrick DNA for cpc560, orig EYFP, opto EYFP, cpc, idiA

‣ Sucrose experiment for cscB 

September 30-October 6

Week 18

‣ RE digest of backbone, psbA2, cpc560, idiA, cscB

‣ Transformation of psbA2, cpc560, idiA, cscB

‣ Inoculation of transformed colonies

‣ Miniprep of inoculation and sequencing

‣ Sucrose assay with cscB

‣ Luciferase assay for cpc, cpc560, psbA2 

October 7-13 

Week 19

‣ Miniprep of transformed BioBrick colonies for opto EYFP, cscB, psbA2, idiA, cpc560

‣ Dried DNA and submitted to iGEM HQ

‣ Started sucrose assay for cscB by inducing with an increased amount of IPTG

‣ Luciferase experiment with high light inducing expression for psbA2, idiA, cpc560, cpc  

October 14-17

Week 20

‣ Sucrose assay experiment for cscB colonies induced with IPTG prior week

‣ Luciferase experiment with 2,2 dipyridyl (iron chelator)

2018 Stony Brook iGEM 

The Stony Brook iGEM Team is proud to present to you their sweet and energy filled project! Made with love <3