Next Generation Synthesis

Joseph Jacobson (MIT)

Recordings

Will be linked after class

Presentations and slides

Will be linked after class

Class assignments

Synthetic oligonucleotides (oligos) enable us to assemble novel DNA sequences or create copies of existing genes. This week's assignment will give you experience building genes and expressing them in bacteria.

Lab Task: Recode green fluorescent protein (GFP) to instead emit blue or cerulean light. Confirm success by transforming your assembled BFP and CFP variants into E coli cells.

Theory Task: Design a gene. Describe a detailed workflow for constructing and expressing it. Identify how the parts of your genetic construct relate to DNA replication and the Central Dogma of Molecular Biology.

Hardware

Function	Item
Polymerase Chain Reaction	Thermocycler
Incubating samples at 37C	Thermocycler, water bath, incubator, or body heat
Separating DNA by size	Gel electrophoresis box and power supply
Visualizing stained DNA	Blue-LED transilluminator
Preparing gel matrix and LB agar plates	Microwave and balance
DNA Extraction from TAE Agarose Gel	Microcentrifuge

Software

Function	Item
Mapping plasmids and recoding the GFP sequence	DNA sequence viewer, such as Benchling, Snapgene, Geneious, ApE, GenBeans, or text editor

Wetware

Function	Item
Amplifying DNA	DNA oligonucleotides and DNA polymerase mix to amplify GFP and pUC19
Golden Gate Assembly	Type IIS restriction enzyme (i.e. BbsI-HF) and T7 DNA Ligase
Setting electrophoresis gel	Tris-acetate-EDTA (TAE) buffer and agarose
Setting agar plates	LB agar dehydrated culture media
Drug selection	Carbenicillin

Oligo Name	Sequence	Oligo Pair Partner	Pair Name
pUC19_GolGat_2026R	CACCACAGAAGACGATCAGCTCACTCAAAGGCGG	pUC19_GolGat_1F	pUC19_GolGat
pUC19_GolGat_1F	CACCACAGAAGACGACGGCATCCGCTTACAGACAA	pUC19_GolGat_2026R	pUC19_GolGat
pGreen_nGFP_GolGat_192R	CACCACAGAAGACGAAGTAGTGACTAGTGTTGGCCA	pGreen_nGFP_GolGat_1F	pGreen_nGFP
pGreen_nGFP_GolGat_1F	CACCACAGAAGACGATAACATGGCTAGCAAAGGAGAAGAACT	pGreen_nGFP_GolGat_192R	pGreen_nGFP
pGreen_cGFP_GolGat_575R	CACCACAGAAGACGAATAGGGCGAATTCGAGCTCG	pGreen_c(G/C/B)FP_GolGat_1F	pGreen_c(G/C/B)FP_GolGat
pGreen_cGFP_GolGat_1F	CACCACAGAAGACGATACTCTGTGCTATGGTGTTCAATGCT	pGreen_cGFP_GolGat_575R	pGreen_cGFP_GolGat
pGreen_cCFP_GolGat_1F	CACCACAGAAGACGATACTCTGACCTGGGGTGTTCAATGCT	pGreen_cGFP_GolGat_575R	pGreen_cCFP_GolGat
pGreen_cBFP_GolGat_1F	CACCACAGAAGACGATACTCTGACCCATGGTGTTCAATGCT	pGreen_cGFP_GolGat_575R	pGreen_cBFP_GolGat
T500_s1	CTATGAGCAAAGCCCGCCGAAAGGCGGGCTTTTCTGTACA	T500_s2	T500
T500_s2	GCCGTGTACAGAAAAGCCCGCCTTTCGGCGGGCTTTGCTC	T500_s1	T500
OR2OR1Pr_s1	CTGATGAGCTAACACCGTGCGTGTTGACAATTTTACCTCTGGCGGTGATAATGGTTGCAG	OR2OR1Pr_s2	OR2OR1Pr
OR2OR1Pr_s2	CAAGCTGCAACCATTATCACCGCCAGAGGTAAAATTGTCAACACGCACGGTGTTAGCTCA	OR2OR1Pr_s1	OR2OR1Pr
5UTR_RBS_s1	CTTGCAATAATTTTGTTTAACTTTAAGAAGGAGATA	5UTR_RBS_s2	5UTR_RBS
5UTR_RBS_s2	GTTATATCTCCTTCTTAAAGTTAAACAAAATTATTG	5UTR_RBS_s1	5UTR_RBS

Brief Protocol

1 Resuspend oligos to make a 100 uM stock and then 10 uM dilutions that combine oligo pairs

Read nmol amount of oligo from tube label
For every nmol, add 10 ul water
Vortex or flick tube and wait several minutes for the oligo to go into solution

2 In a separate tube combine 80 ul water and 10 ul of each 100 uM oligo in a pair to make 10 uM paired dilutions

Oligo pairs that will be annealed as duplexes are named with the format X_s1 and X_s2. As an example, for X=T500:

Item	Vol to Mix
T500_s1	10 ul
T500_s2	10 ul
Water	80 ul

Oligo pairs that will be used together as PCR primers together are named with the format XGolgat_1F and X_Golgat##R or XY_Golgat_1F and X_Y'_Golgat##R.

3 Prepare Golden Gate annealed duplex fragments.

In separate PCR tubes, mix the following

Item	Vol to Mix
X_s1 + X_s2 dilution (10 uM)	8 ul
T4 DNA ligase buffer (10x)	1 ul
T4 Polynucleotide Kinase (T4 PNK)	1 ul

Incubate mix at 37C for 1 hr
Heat mixtures at 95C in thermocycler for 5 min and gradually cool to RT for 1 hr

4 Prepare the other Golden Gate fragments by PCR from plasmid X in primer pair X_Y_Golgat.

In separate PCR tubes, mix the following 50 ul PCR reactions

Item	Vol to Mix
X_Y_GolGat primer pair (10 uM)	2.5 ul
Template plasmid X	1 ul
dNTPS (10 uM)	1 ul
Phusion polymerase buffer (5x)	10 ul
Phusion DNA polymerase	1 ul
Water	34.5 ul

Run the following PCR program in your thermocycler with the lid set to 105 C

Stage	Steps
1	Initial denaturation: 98C for 30 sec
2 (30x cycles)	1. Denaturation: 98C for 10 sec, 2. Annealing: 60C for 30 sec, 3. Extension: 72C for 30 sec/kb
3	Final extension: 72C for 5 min

Run finished PCR reaction on a 1% agarose TAE gel (see protocol homework 2)
Purify the correct-sized band with a gel extraction kit (see kit manufacturer’s protocol)

5 Combine and assemble the Golden Gate fragments

For each fragment from step 4 derived from pGreen_c(G/C/B)FP_GolGat_1F, mix the following in a thermocycler tube

Item	Vol to Mix
6 fragments from steps 3 and 4	1 ul each
Cutsmart buffer (10x)	2 ul
ATP (10 mM)	2 ul
T7 or T4 DNA ligase	1 ul
BbsI-HF	1 ul
Water	8 ul

Run the following assembly program in your thermocycler with the lid set to 105 C

Stage	Steps
1 (30x cycles)	1. Digest: 37C for 5 min, 2. Ligate: 16C for 5 min
2	Heat denature: 55C for 10 min

Next generation Synthesis