Team:HK SSC/Contribution
Contribution
Documentation of Neutral Sites
Our engineering design involves using CRISPR technology to edit the chromosome of S.elongatus UTEX 2973. crRNA complementary to neutral sites in the chromosome of S.elongatus UTEX 2973 are needed. Neutral sites are sequences in the chromosome that do not affect the organism’s phenotype when altered, and therefore are commonly used for genomic editing sites1. We noticed how the documentation about Neutral Site I and Neutral Site II in the chromosome of S.elongatus in parts BBa_K3228020, BBa_K3228021, BBa_K3228023, and BBa_K3228024 by iGEM team Marburg 2019 was brief and so we added information learned from literature and citations on their main and design pages. More thorough documentation of the neutral sites in the chromosome of S.elongatus UTEX 2973 can help future teams design engineering tools used at the site to more efficiently engineer the organism.
Generating a Codon Usage Table for S.elongatus UTEX 2973
With reference to the data analysis method used by Rajneesh et al.(2017), we generated a codon usage table of S.elongatus UTEX 2973 by ourselves since we could not find it elsewhere. We used ATGme to codon optimize and silent mutate our gene fragments, the codon usage table in the PDF below can be directly copied and pasted into the blank space provided in the webpage. We also used FAIR to check for internal repeats that would make DNA synthesis difficult and silent mutated them.
|
Codon |
AA |
RSCU |
Codon |
AA |
RSCU |
|
TTT |
F |
0.9402 |
GCT |
A |
0.8920 |
|
TTC |
|
1.0598 |
GCC |
|
1.5488 |
|
TTA |
L |
0.3057 |
GCA |
|
0.2787 � |
|
TTG |
|
2.8540 |
GCG |
|
1.2803 |
|
CTT |
|
0.4983 |
TAT |
Y |
0.4335 |
|
CTC |
|
0.6773 |
TAC |
|
1.2332 |
|
CTA |
|
0.1167 |
CAT |
H |
1.4185 |
|
CTG |
|
1.5482 |
CAC |
|
0.5815 |
|
ATT |
I |
1.0768 |
CAA |
Q |
1.2755 |
|
ATC |
|
1.2115 |
CAG |
|
0.7245 |
|
ATA |
|
0.7117 |
AAT |
N |
1.4530 |
|
GTT |
V |
1.0822 |
AAC |
|
0.5470 |
|
GTC |
|
1.1438 |
AAA |
K |
1.6078 |
|
GTA |
|
0.7168 |
AAG |
|
0.3922 |
|
GTG |
|
1.0565 |
GAT |
D |
1.6057 |
|
TCT |
S |
0.6562 |
GAC |
|
0.3943 |
|
TCC |
|
0.8173 |
GAA |
E |
0.8272 |
|
TCA |
|
0.0977 |
GAG |
|
1.1728 |
|
TCG |
|
0.6280 |
TGT |
C |
0.7152 |
|
AGT |
|
1.0898 |
TGC |
|
1.2848 |
|
AGC |
|
2.7112 |
CGT |
R |
0.7622 |
|
CCT |
P |
0.2115 |
CGC |
|
1.7628 |
|
CCC |
|
0.9025 |
CGA |
|
0.478 |
|
CCA |
|
2.1568 |
CGG |
|
0.966 |
|
CCG |
|
0.7292 |
AGA |
|
1.0065 |
|
ACT |
T |
1.4242 |
AGG |
|
1.0243 |
|
ACC |
|
1.4323 |
GGT |
G |
0.8857 |
|
ACA |
|
0.4442 |
GGC |
|
1.5378 |
|
ACG |
|
0.6993 |
GGA |
|
0.2000 |
|
|
|
GGG |
|
1.3763 |
The codons displayed in bold are the preferred codon(s)
Protocol for Electroporation of S.elongatus UTEX 2973
When researching the gene manipulation of S.elongatus UTEX 2973, we realized there is minimal documentation of the method of electrotransformation. Using work by Mühlenhoff and Chauvat (1996) as a reference, we devised a protocol for transformation by electroporation of S.elongatus UTEX 2973 cells. The practicability of the protocol is confirmed by the resistance shown to kanamycin by S.elongatus UTEX 2973 cells transformed with panS-loxP-MCS, which harbors a kanamycin resistance cassette, in our project. The protocol can be found here.
References:
- Ruffing, A. M., Jensen, T. J., & Strickland, L. M. (2016). Genetic tools for advancement of Synechococcus sp. PCC 7002 as a cyanobacterial chassis. Microbial Cell Factories, 15(1). https://doi.org/10.1186/s12934-016-0584-6
- Rajneesh, Pathak, J., Kannaujiya, V. K., Singh, S. P., & Sinha, R. P. (2017). Codon usage analysis of photolyase encoding genes of cyanobacteria inhabiting diverse habitats. 3 Biotech, 7(3). https://doi.org/10.1007/s13205-017-0826-2
- Mühlenhoff, U., & Chauvat, F. (1996). Gene transfer and manipulation in the thermophilic cyanobacteriumSynechococcus elongatus. Molecular and General Genetics MGG, 252(1–2), 93–100. https://doi.org/10.1007/bf02173209